Research Article
Received: 5 June 2009
Accepted: 9 August 2009
Published online in Wiley Interscience: 29 September 2009
(www.interscience.wiley.com) DOI 10.1002/jrs.2483
Confocal Raman microscopy for simultaneous
monitoring of partitioning and disordering
of tricyclic antidepressants in phospholipid
vesicle membranes
Christopher B. Foxa and Joel M. Harrisa,b∗
Characterization of drug–membrane interactions is important in order to understand the mechanisms of action of drugs
and to design more effective drugs and delivery vehicles. Raman spectra provide compositional and conformational
information of drugs and lipid membranes, respectively, allowing membrane disordering effects and drug partitioning to
be assessed. Traditional Raman spectroscopy and other widely used bioanalytical techniques such as differential scanning
calorimetry (DSC) and nuclear magnetic resonance (NMR) typically require high sample concentrations. Here, we describe how
temperature-controlled, optical-trapping confocal Raman microscopy facilitates the analysis of drug–membrane interactions
using micromolar concentrations of drug, while avoiding drug depletion from solution by working at even lower lipid
concentrations. The potential for confocal Raman microscopy as an effective bioanalytical tool is illustrated using tricyclic
antidepressants (TCAs), which are cationic amphiphilic molecules that bind to phospholipid membranes and influence lipid
phase transitions. The interaction of these drugs with vesicle membranes of differing head-group charge is investigated while
varying the ring and side-chain structure of the drug. Changes in membrane structure are observed in Raman bands that report
intra- and intermolecular order versus temperature. The partitioning of drugs into the membrane can also be determined from
the Raman scattering intensities. These results demonstrate the usefulness of confocal Raman microscopy for the analysis
of drug–membrane systems at biologically relevant drug concentrations. Effective tools for monitoring drug–membrane
c 2009 John Wiley & Sons, Ltd.
interactions are crucial for rational design of new drugs. Copyright Keywords: confocal Raman microscopy; drug–membrane interactions; tricyclic antidepressants; phospholipid vesicles
Introduction
498
Drug–membrane interactions constitute an essential factor in
understanding the effects of drugs and improving their design.
Drugs interact with membrane assemblies in multiple ways,
whether they are being transported through a membrane,
binding to proteins in the membrane, acting directly on the
phospholipid molecules themselves, or being carried in liposomes.
Furthermore, the cooperative nature of protein–membrane
interactions means that protein function is strongly influenced
by the membrane bilayer phase or structure. Thus, numerous
techniques have been employed to elucidate drug–membrane
activity and the influence of drugs on lipid phase transition
behavior,[1,2] including differential scanning calorimetry (DSC),[3 – 8]
vibrational spectroscopy,[5,7 – 9] X-ray diffraction (XRD),[10] nuclear
magnetic resonance (NMR),[10,11] fluorescence spectroscopy,[3]
and molecular dynamics simulations.[12 – 15] Despite the variety
of methods employed to study lipid phase transitions and
drug–membrane interactions, there are major drawbacks with
many of these methods: for high sensitivity methods such as
fluorescence, there is a need for labeling; the influence of the
fluorescent probe on the structure and behavior of the system
must be considered.[16,17] With less sensitive methods such as DSC,
NMR, XRD, or traditional infrared and Raman spectroscopies, high
drug and lipid concentrations are required, typically in the range
of 10–100 mM.[5,18,19] While Raman scattering is a weak effect,
this technique is well suited to study phospholipid structure; the
J. Raman Spectrosc. 2010, 41, 498–507
method requires no labeling and is compatible with aqueous
buffers, requiring a minimum of sample preparation. When
integrated with a confocal microscope, the efficiency of Raman
spectroscopy can be greatly improved for investigating small
colloidal structures[20 – 22] including lipid vesicles or liposomes.[23]
With a focused excitation laser beam in a confocal Raman
microscope, it is possible to optically trap individual vesicles so
that Raman scattering is collected from a small (<1.5 fl) volume
dominated by the trapped vesicle,[23] such that contributions from
the surrounding solution are minimized.[24] With modest incident
lasers powers of a few milliwatts, the confocal Raman microscope
allows lipid vesicles to be trapped indefinitely in the laser focus
and their spectra to be acquired over long observation times
while conditions are varied. The partitioning of drugs in a lipid
membrane can be quantified in this experiment, from the Raman
scattering intensity of the drugs that accumulate in the lipid bilayer
of the trapped vesicle.[25] Changes in lipid membrane structure
due to its interactions with drugs or other small molecules can be
characterized by relative Raman peak intensities that respond to
∗
Correspondence to: Joel M. Harris, Department of Chemistry, University of Utah,
315 South 1400 East, Salt Lake City, UT 84112-0850, USA.
E-mail: [email protected]
a Department of Bioengineering, University of Utah, Salt Lake City, UT, USA
b Department of Chemistry, University of Utah, Salt Lake City, UT, USA
c 2009 John Wiley & Sons, Ltd.
Copyright Confocal Raman microscopy of tricyclic antidepressants in vesicle membranes
J. Raman Spectrosc. 2010, 41, 498–507
Materials and Methods
Reagents and materials
DPPC and DPPG of >99% purity were purchased from Avanti Polar
Lipids (Alabaster, AL). Amitriptyline hydrochloride, nortriptyline
hydrochloride, and protriptyline hydrochloride were obtained
from Sigma (St. Louis, MO). An extruder was purchased from
Avanti Polar Lipids. Track-Etch polycarbonate membranes were
made by Nucleopore (Pleasanton, CA). Water was quartz-distilled
and then filtered with a Barnstead (Boston, MA) NANOpure II and
had a minimum resistivity of 18.0 M cm. Buffer components were
supplied by Mallinckrodt (Paris, KY).
Sample preparation
One milligram of phospholipid (Tm 41 ◦ C) in chloroform was
pipetted into a 15-ml glass vial and dried in vacuum for at least
3 h to remove the solvent. The film was hydrated with 1 ml
of buffer above Tm and stored at ∼3 ◦ C. Lipid solutions were
used within 1 week of initial hydration. When removed from the
refrigerator for use, lipid solutions were incubated for at least
1 h at a temperature above the Tm . In the case of pure DPPC,
vesicles were prepared by extruding the hydrated solution, at
temperatures 10–20◦ above the Tm of the phospholipid, 15 times
through a polycarbonate filter having pore openings of 1 µm
diameter. DPPG forms vesicles spontaneously, so that extrusion
was not necessary. The vesicles dispersions were diluted 500-fold
into solutions containing 50 mM phosphate buffer (pH 7.2). A 40-µl
aliquot of this solution was pipetted into a small-volume copper
sample cell having a UV-ozone-cleaned coverslip window adhered
to its base.[40 – 46,48,49,51,52] Vesicles collected in the optical trap were
either individual or small aggregates of ∼2–8 vesicles; both types
of samples were observed, and no structural differences between
membranes were detected. Since vesicles containing DPPG were
not extruded, a range of vesicle sizes was present. An effort was
made to analyze vesicles near 1 µm diameter; however, larger
vesicles up to 4 µm in diameter were also trapped when smaller
vesicles could not be found.
Temperature-dependent Raman microscopy
The temperature-dependent Raman microscope setup has been
described previously.[37] Briefly, the beam from a 647.1-nm krypton
ion laser was passed through a band-pass filter and a 4× beam
expander mounted on a Nikon TE 300 inverted fluorescence
microscope and then through another band-pass filter. The
expanded laser beam was then reflected by a dichroic beam
splitter into a 100× oil immersion 1.4 NA microscope objective
where it was focused to a 0.6-µm-diameter spot inside the sample
cell. The same objective was used to collect the Raman scattering
from the sample, which was passed through the beam splitter,
then through two high-pass filters, and then focused on to a
monochromator with 50 µm slit width. A charge-coupled device
(CCD) camera was employed to collect the spectra over three pixels
or 78 µm in the vertical dimension. The spectral resolution was
6 cm−1 . Laser power at the sample was ∼23 mW and integration
time was 1 min. In general, three samples at each data point were
collected; however, some spectra were deleted because of low
signal (ambiguous results) or because vesicles were dislodged
from the optical trap as a result of air bubbles formed during the
temperature ramp. White light correction and baseline fitting of
c 2009 John Wiley & Sons, Ltd.
Copyright www.interscience.wiley.com/journal/jrs
499
changes in intra- and intermolecular order.[5,7,9,25 – 35] In a confocal
Raman microscopy experiment, these structural changes in the
membrane can be combined with quantitative information on the
degree of drug partitioning,[25] yielding a detailed picture of the
drug–membrane interactions. An important advantage of opticaltrapping confocal Raman microscopy for such studies is that
membrane effects of very low, clinically relevant concentrations
of drug can be investigated. Samples are typically prepared with
a few micromolar lipid concentrations, which yield a sufficient
number density of vesicles that is relatively easy to locate and trap
a vesicle for study. This low lipid concentration and corresponding
small membrane volume avoids depleting clinically relevant (µM)
drug concentrations through membrane partitioning, which is a
significant limitation of the less sensitive methods that require
10 mM or higher lipid concentrations.
Lipid membrane assemblies undergo characteristic thermal
phase transitions,[30,32,35,36] which are characterized by changes in
lipid structural order. Small-molecule drugs most often decrease
and widen the main lipid phase transition (Tm ) depending
on drug concentration,[37] but may also increase Tm if they
enhance lipid packing.[27,38,39] Lipid phase transitions can be
detected in confocal Raman microscopy by the use of a
temperature-controlled stage and metal sample cell, allowing
sample temperatures to be varied from ∼5 to 55 ◦ C while requiring
only small sample volumes (<40 µl).[37] While temperaturecontrolled confocal Raman microscopy has been used to study
membrane structural changes in phase transitions of individual
vesicle membranes prepared from pure synthetic lipids,[35] the
technique has not been applied to the study of the effects of
drugs on the structure and phase transitions of lipid membranes,
where both the drug partitioning as a function of temperature and
membrane phase transition can simultaneously be investigated.
In this study, the partitioning of tricyclic antidepressants (TCAs)
into both zwitterionic and negatively charged phospholipid
membranes and their influence on the membrane thermal phase
transitions are investigated using optical-trapping confocal Raman
microscopy. Tricyclic antidepressants are cationic, amphiphilic,
small-molecule drugs; the mechanism of action of TCAs is not
fully understood, but it is clear that they bind to phospholipid
membranes.[2] TCA–membrane interactions are critical because
TCAs are known to induce lipidosis, or intracellular accumulation
of lipids.[40 – 49] Literature reports on the activity of TCAs indicate
strong but varying membrane interactions depending on the
particular drug and lipid structure, including effects on lipid phase
transitions.[49,50] The TCAs chosen for this study have structural
variations in both the ring and side-chain structure. With similar
pKa values ranging from ∼9.4 to 10.0, the drugs have essentially
the same charge state (+1) at the experimental pH of 7.2.
Variations in their membrane activity are explored in terms of the
structural differences of the drugs or variations in lipid headgroup
structure. Vesicles composed of 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (DPPG) in solutions containing varying
concentrations of TCAs are monitored by confocal Raman
microscopy as a function of temperature in order to determine the
drug partitioning and disordering behavior. The utility of confocal
Raman microscopy for investigating low concentrations of drug in
drug–membrane studies is discussed.
C. B. Fox and J. M. Harris
the Raman spectra with a fourth-order polynomial function were
done in Matlab (MathWorks), as described previously.[37]
A water-cooled Peltier stage (Technical Video, Ltd.) was
integrated with a copper block to surround the sample cell and
allow efficient heat transfer.[23] Temperature measurements were
collected from a well in the copper block using a thermocouple.
Temperature was changed at a rate of approximately 1 ◦ C/min,
and reported values were averaged from recorded temperatures
after each 1-min signal integration. All peak intensities were
obtained from the maximum peak intensity value in the region
near the indicated wavenumbers. The peak ratio from the C–H
stretching region, where scattering from the terminal methyl C–H
symmetric stretching mode (2934 cm−1 ) is ratioed to the scattering
of the C–H asymmetric stretching mode (2883 cm−1 ), is plotted
versus temperature to monitor the membrane phase transition. A
smoothed first derivative of the temperature-dependent ratios
is computed with a five-point second-order Savitzky–Golay
filter,[37] where the peak of the numerical derivative reports
the inflection point of the melting transition. Drug partitioning
data were plotted using the intensity of the C C ring stretch
of the drug (1600 cm−1 ) ratioed to the C O stretching mode
(∼1730 cm−1 ) of the lipid, since the latter was not observed to
change significantly with temperature. The scattering intensities
of the C C ring stretch of all three drugs, as 200 mM standard
solutions prepared in methanol, were measured and compared.
The intensities of amitriptyline and nortriptyline were equivalent,
while the scattering from protriptyline was threefold greater, likely
due to the greater polarizability of the conjugated double bond
in the central ring of this compound. The observed partitioning
ratio of this drug was divided by 3 to account for its greater
Raman scattering cross section. The temperature-dependent drug
partitioning ratios were smoothed using a three-point moving
average. In general, the ‘below Tm ’ drug partitioning values were
taken 6 ◦ C below the measured Tm of the lipid sample and the
‘above Tm ’ drug partitioning ratios were taken 6 ◦ C above the
measured Tm , although some samples did not span this entire
temperature range. For these cases, the closest data point was
used as long as it was below or above the Tm . The peak ratio
I1600 /I1730 from control lipid samples was subtracted from the
drug-containing samples.
Self-modeling curve resolution (SMCR) analysis of temperaturedependent Raman spectra from lipid samples has been described
in a previous publication.[53] Briefly, this method projects the
Raman spectra into eigenvector space, eliminating eigenvectors
that represent random noise, and rotating the minimal basis set of
eigenvectors, representing correlation in the data, back into real
space by assuming non-negativity in pure component spectra and
their temperature profiles. SMCR is used here to compare the pure
component spectra representing the lipid fluid phase of control
lipid samples and lipid samples containing drug.
Results
500
The structures of the tricyclic antidepressant drugs amitriptyline, nortriptyline, and protriptyline, and their octanol/water and
liposome/water partition coefficients[35] are shown in Fig. 1. The
liposome formulation used to determine the liposome/water partition coefficients consisted of DPPC (46%) : DPPG (24%) : cholesterol
(30%).[47,54]
The drug structures differ in the number of methyl groups at the
end of the aliphatic chain and the location of the C C bond that
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is found in the center ring or on the aliphatic chain. The structures
and phase transition temperatures of the 16-carbon acyl chain
lipids DPPC (zwitterionic) and DPPG (anionic) are also presented
in Fig. 1.
The Raman spectrum of an optically trapped DPPC vesicle with
peak assignments is shown in Fig. 2, where it is compared to
Raman scattering from pure DPPG and DPPC : DPPG 1 : 1 molar
ratio vesicles at room temperature. The Raman scattering from
samples of pure DPPC vesicles was typically more intense than that
of vesicles of DPPG; there is a greater likelihood of DPPC vesicles
being multilamellar (more than one lipid bilayer), while DPPG
vesicles are typically unilamellar due to electrostatic repulsion
between the charged head groups.[47] Spectra were collected
continuously for 1 min integration times as the temperature was
slowly changed (∼1 ◦ C/min), and contained information indicative
of changes in the lipid structural order.
By monitoring changes in peak intensities and wavenumbers
of Raman scattering from phospholipid vesicles, differences in
acyl chain conformation and the corresponding intra- and intermolecular membrane order can be determined.[5,7,9,26 – 31] Phase
changes in phospholipid assemblies can be detected in either
the C–C stretching (∼1060–1120 cm−1 ) or the C–H stretching
(∼2800–3100 cm−1 ) regions of the spectrum. The methylene C–C
stretching region reflects the evolution from trans (∼1065 and
1114 cm−1 ) to gauche (∼1086 cm−1 ) conformations in the lipid
acyl chains as a membrane becomes more disordered.[7,31,55] The
methylene C–H stretching region also reflects membrane order
and consists of three strong bands that represent C–H symmetric stretching (∼2845 cm−1 ), C–H asymmetric stretching (∼2883
cm−1 ), and terminal methyl C–H symmetric stretching (∼2934
cm−1 ). Despite overlap between these bands, the peak intensity
ratio for the 2934 to 2883 cm−1 bands correlates with changes in
interchain interactions as well as intramolecular conformations as
the membrane becomes disordered.[5,9,31]
The effect of increasing amounts of nortriptyline on the lipid
main phase transition profile of DPPC, DPPG, and DPPC : DPPG 1 : 1
is shown in Fig. 3 by monitoring the change in the C–H stretching
peak ratio I2934 /I2883 with temperature. The drug decreases Tm and
widens the transition profile width. Similar effects were noticed
for the other TCAs, protriptyline and amitriptyline. The Tm value
for each sample is taken as the inflection point on the slope of the
I2934 /I2883 curve and is calculated using a 5-point Savitzky–Golay
filter as described in the Experimental section. In Figs 4 and 5, the
effects of the TCAs on lipid Tm values are plotted according to drug
concentration. The influence of the drugs on different lipids is
apparent. However, when acting on the same lipid, no significant
difference is seen between the disordering effects of the three
drugs. Table 1 presents the resulting Tm values for comparison.
Interestingly, the disordering effects of TCAs, while obviously
influencing Tm , do not appear to increase overall lipid disorder
once the fluid phase is reached. This is observed by comparing the
pure component spectra obtained by SMCR representing the fluid
phase DPPC membrane both with and without drug in Fig. 6.
The temperature-dependent partitioning of the drugs is
apparent in Fig. 7(a), where a Raman spectrum from a vesicle
sample in a 500 µM amitriptyline solution at low temperature
is compared with one at high temperature. In Fig. 7(b), the
Raman scattering peak representing the C–C ring stretch of
the drug (1600 cm−1 )[25,56] is ratioed to the C O stretch of
the lipid (∼1730 cm−1 ). In DPPC, the drug concentration in the
membrane significantly increases near the Tm , revealing that the
drug preferentially partitions into the fluid phase membrane. This
c 2009 John Wiley & Sons, Ltd.
Copyright J. Raman Spectrosc. 2010, 41, 498–507
Confocal Raman microscopy of tricyclic antidepressants in vesicle membranes
Figure 1. Structures of the tricyclic antidepressant drugs and phospholipid molecules. Reported drug partition coefficients[47] and lipid phase transition
temperatures[47,54] are also shown.
500
C-C stretch
(gauche)
C-H antisymmetric stretch
C-H symmetric stretch
CH2 twist
CH2 bend
C-C stretch
(trans)
400
Raman Intensity / 10 PE
terminal CH3 symmetric stretch
C-C stretch (trans)
C-N symmetric
stretch
DPPC
300
1 min integration
C=O stretch
200
DPPG
100
DPPC:DPPG 1:1
0
600
900
1200
1500
1800
2100
2400
2700
3000
Wavenumber / cm-1
Figure 2. Raman spectra and peak assignments of optically trapped
phospholipid vesicles. DPPC is compared to DPPG and DPPC : DPPG 1 : 1
molar ratio vesicles.
temperature-dependent partitioning is seen in the other lipid
samples as well, but it is usually less dramatic and sometimes
not apparent at all (Fig. 8). Moreover, the lipid samples containing
DPPG generally contain much more drug at all temperatures
compared to DPPC, presumably due to electrostatic interactions
(see the section on Discussion).
Discussion
Membrane disordering effects of TCAs
J. Raman Spectrosc. 2010, 41, 498–507
c 2009 John Wiley & Sons, Ltd.
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501
The application of optical-trapping confocal Raman microscopy for
the elucidation of several aspects of drug–membrane interactions
has been developed here using TCAs. TCAs provide an intriguing
model for the study of the influence of cationic amphiphiles of
varying structure in different lipid environments. Small-molecule
drugs with membrane affinity can have varying effects on
phospholipid vesicles. Small organic molecules have been shown
to increase lipid order (higher Tm ) or disrupt lipid order (lower Tm ),
depending on how the structure of the molecule intercalates into
the lipid bilayer.[57]
The first result apparent from Figs 3–5 is that TCAs have
disordering effects on lipid bilayers, both neutral and anionic, as
manifested by the decreased Tm values. This indicates the manner
of drug packing in the lipid bilayer. Small molecules that increase
lipid order tend to fit neatly into the gaps between lipid chains,[58]
but TCAs are sufficiently bulky to create steric disruption. Indeed,
an NMR study has shown that the TCAs are probably located with
their cyclic rings near the acyl chains and their polar carbon tail
‘snorkeling’ up near the lipid headgroup; in other words, TCAs are
mostly in the lipid interfacial/headgroup region, disrupting lipid
packing and providing the acyl chains with more room to move.[59]
In general, the literature suggests that TCAs are located with the
rings near the lipid acyl chains and the charged tail near the
lipid headgroup,[60] although one report concludes that the drug
conformation in DPPC is quite mobile but preferentially assumes
a state where the charged tail is folded back on the rings.[49,51,61]
Indeed, ring flexibility in TCA differs depending on specific drug
structure.[42] The results in Figs 3–5 align well with the general
picture from the literature; the fact that the drugs lower the lipid
Tm means that they must affect the acyl chain packing in the lipid.
Also, evidence for electrostatic interactions is clear: the drug has
greater disordering effects in anionic membranes. Moreover, the
partitioning of the TCAs shown in Fig. 8 reveals that more drug is
present in anionic membranes, as discussed in more detail below.
Figures 3–5 and Table 1 clearly show that TCAs have a greater
effect on lipid order in DPPG compared to DPPC as manifested
by the lipid Tm values at different drug concentrations. This
finding is in accordance with another study that examined TCA
effects on DPPC and DPPG at higher drug concentrations,[62] but
contradicts an earlier report that examined the effects of several
different cationic amphiphilic drugs with varying phospholipid
molecules.[45] In this earlier report, it was found that at very
C. B. Fox and J. M. Harris
1.0
0 µM drug (pure lipid)
20 µM drug
100 µM drug
500 µM drug
0.9
DPPC
DPPG
DPPC:DPPG 1:1
46
44
42
0.8
40
0.7
38
0.6
36
DPPC
34
0.5
32
0.4
30
1.0
I2934/I2883
28
0.9
46
0.8
44
42
0.7
40
0.6
Tm/ °C
DPPG
0.5
38
36
34
0.4
32
1.0
30
0.9
28
0.8
46
0.7
44
42
0.6
40
DPPC:DPPG 1:1
0.5
38
36
0.4
20
25
30
35
40
45
50
55
34
32
Temperature /°C
Figure 3. Effects of increasing concentrations of nortriptyline on the phase
transition profiles of DPPC (top), DPPG (middle), and DPPC : DPPG 1 : 1
(bottom) as revealed by the I2934 /I2883 lipid peak ratio.
30
28
0
502
high (mM) drug concentrations, the drug overcomes the influence
of the phospholipid headgroup, resulting in similar Tm values for
DPPA (1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid), DPPG, and
DPPC, even though the pure lipids have different Tm values.[48] The
depressions in Tm values for DPPC and DPPG shown in Figs 4 and
5 are in good agreement to values obtained from fluorescence
studies using higher drug concentrations,[48] suggesting that a
plateau in lipid disordering is reached. This plateau effect has
been reported for other cationic amphiphilic drugs in DPPG.[45]
Indeed, in Figs 4 and 5, the Tm of DPPG values show a plateau
effect, where 20–100 µM drug shows similar disordering behavior
as 500 µM drug. However, the decrease in the Tm of DPPC follows
a continuous decrease with increasing drug concentration and
no apparent plateau, perhaps because the effective ‘plateau’
concentration is higher than the concentrations studied here
(≤500 µM). This difference in TCA plateau behavior effects between
DPPC and DPPG could arise because the attraction of the drug
to zwitterionic DPPC derives from relatively weak hydrophobic
interactions, whereas the interaction of the drug with anionic DPPG
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100
200
300
400
500
Drug concentration / µM
Figure 4. Effects of increasing concentrations of protriptyline (top),
amitriptyline (middle), and nortriptyline (bottom) on lipid Tm values.
includes a strong electrostatic attraction that quickly saturates the
lipid headgroups with the accumulation of drug.[63] It has been
found that electrostatic interactions with cationic drugs dominate
in mixtures of neutral and anionic lipids.[49] In addition, another TCA
drug (chlorpromazine) was shown to have a greater disordering
effect on DPPG than DPPC monolayers; this effect was shown by
IR spectroscopy to be in the headgroup region instead of the acyl
chains.[44,49] Finally, while chlorpromazine was found to lower the
Tm of neutral as well as anionic membranes, greater disrupting
effects were observed using NMR in the anionic membrane.[64]
In short, while there is disagreement about the extent of the
disordering effects of TCAs in lipids, it is clear that anionic lipids
are more strongly affected than zwitterionic lipids.
c 2009 John Wiley & Sons, Ltd.
Copyright J. Raman Spectrosc. 2010, 41, 498–507
Confocal Raman microscopy of tricyclic antidepressants in vesicle membranes
46
Protriptyline
Amitriptyline
Nortriptyline
45
44
43
42
41
40
39
DPPC
38
42
40
Tm/ °C
38
36
34
32
30
28
DPPG
26
46
44
activity), but there was no apparent difference between amitriptyline and nortriptyline.[43] In an analogous
system to amitriptyline and nortriptyline, an NMR study concluded that the extra terminal methyl group in imipramine has
more of a lipid headgroup disordering effect than desipramine
in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-1palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-sn-glycerol) (POPG)
POPG mixture, probably due to steric effects; however, it is also
noted that these differences may not be directly biologically interpretable since desipramine is, ironically, more prone to induce
lipidosis than imipramine.[61] Finally, one study concluded that the
aliphatic tail present on TCAs is essential for their effects on neutral
lipid membranes since iminodibenzyl, which lacks the hydrocarbon tail, showed minimal effects.[51] Therefore, while some reports
indicate that an extra terminal methyl group leads to more greater
disordering, this finding is not conclusive and is not apparent from
the results in Figs 4 and 5 and Table 1.
It is also interesting to note that the level of disorder in control
lipid samples due to temperature is not significantly increased
when drug is present. In other words, while the presence of drug
decreases the main phase transition temperature (destabilizing
the gel phase), the extent of lipid disorder reached in the final
fluid phase does not significantly increase with the presence of
drug. This is apparent in Fig. 3, where the peak intensity ratio
representing lipid disorder in drug solutions is not significantly
greater than control lipid samples once the fluid phase is
reached. Other Raman scattering peak intensities also appear
similar to control lipid samples, as revealed in comparing the
pure component spectra obtained from SMCR representing the
fluid-phase DPPC membrane with or without drug (Fig. 6).
42
40
Drug partitioning between membrane and solution
38
36
34
32
30
DPPC:DPPG 1:1
28
0
100
200
300
400
500
Drug concentration / µM
Figure 5. Comparison of the effects of each of the TCA drugs on DPPC
(top), DPPG (middle), and DPPC : DPPG 1 : 1 (bottom) Tm values.
J. Raman Spectrosc. 2010, 41, 498–507
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503
Studies correlating the structure of different TCAs to lipid membrane disordering effects have come to different conclusions. For
instance, using a fluorescence depolarization membrane probe,
nortriptyline and protriptyline were observed to have similar disordering effects on lipid Tm in mixed liposomes composed of DPPC
and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), but no effect could be seen in pure DPPC liposomes at 100 µM drug.[65] The
TCAs amitriptyline, nortriptyline, imipramine, and desipramine
were shown by calorimetry to have similarly lowered dipalmitoyllecithin lipid Tm at 1 : 1 drug : lipid molar ratios, but the extra
methyl group on imipramine and amitriptyline caused a multiphase thermal profile.[66] In contrast, another fluorescence study
found that amitriptyline and nortriptyline had a greater lipid disordering effect than imipramine and desipramine in 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC), DMPC/cholesterol, and
synaptosomal membranes (including effects on ATPase
Drug partitioning between membrane and solution is an important
parameter in its pharmacological function. For instance, it has
been found that membrane/plasma partitioning values for TCAs
are correlated to drug concentrations in brain homogenate.[43]
Several interesting conclusions can be drawn from Figs 7 and
8. In Fig. 8, it apparent that each of the drugs partitions
preferentially in membranes containing DPPG, confirming that
electrostatic interactions play a significant role in the membrane
affinity of the drug molecules. This is an important conclusion
since biological lipid assemblies consist of a multiplicity of
lipid molecules including charged, neutral, saturated acyl chains,
unsaturated acyl chains, and headgroup modifications.[67,68] In the
case of brain tissue, where antidepressants are designed to have
significant effects, anionic lipids (including phosphatidylglycerols)
form a major component of the lipid content.[39] In fact, lipid
composition has been shown to be important in mental disorders,
and antidepressants can alter this membrane lipid content.[69]
Therefore, the effect of TCAs on different lipid compositions is
integral to understanding their biological functions, and it is
apparent from Fig. 8 that anionic lipids are important for increased
TCA affinity for lipid membranes.
An additional conclusion that can be drawn from Figs 7 and 8
is that TCA partitioning into the membrane increases significantly
above the lipid Tm compared to below Tm . In other words, TCAs are
able to bind much more readily to fluid-phase lipids, as suggested
by previous studies.[70] The TCA chlorpromazine was found to have
a ∼3.5 times higher affinity for egg phosphatidylcholine (eggPC)
and egg phosphatidic acid (eggPA) monolayers compared to
the pure lipids, presumably due to more disorder in the egg
C. B. Fox and J. M. Harris
Table 1. Lipid phase transition temperatures influenced by tricyclic antidepressant drugsa
0 µM prot
0 µM ami
0 µM nor
20 µM prot
20 µM ami
20 µM nor
100 µM prot
100 µM ami
100 µM nor
500 µM prot
500 µM ami
500 µM nor
a
DPPC
(◦ C)
σ
No.
samples
DPPG
(◦ C)
43.43
44.13
42.7
43.2
44.08
42.53
41.9
41.87
41.83
39.93
39.17
39.93
0.34
0.75
0.31
1.15
1.24
0.46
0.38
1.09
0.21
0.86
0.69
0.86
3
3
3
3
2
3
3
3
3
3
3
3
41.65
39.9
40.43
33.7
34.95
31.98
30.7
32.13
31.62
29.6
31.2
32.05
σ
1.67
1.16
1.12
2.91
1.05
0.69
2.02
1.46
0.83
4.83
No.
samples
DPPC : DPPG
(◦ C)
σ
No.
samples
1
3
3
3
3
3
3
3
3
3
1
3
41.33
43.53
39.55
40.48
39.78
36.55
35.8
40.28
38.35
33.45
34.53
30.78
1.72
2.16
1.56
0.39
0.39
0.99
0.07
0.75
2.26
4.12
1.1
0.11
3
3
2
2
2
2
2
3
2
3
2
2
prot, ami, and nor refer to protriptyline, nortriptyline, and amitriptyline, respectively.
Control DPPC
Raman Intensity
DPPC in 500µM amitriptyline
1000
1100
1200
1300
1400
1500
2900 3000 3100
Wavenumber / cm-1
Figure 6. Fluid-phase spectra of DPPC vesicle samples in buffer without
drug (solid line) or in buffer with 500 µM amitriptyline (dotted line) obtained
by SMCR indicating no significant difference in lipid disorder.
504
lipids.[67,71 – 74] This affinity for fluid phase membranes most likely
is because the ordered gel phase lipid membrane exhibits a high
enthalpy penalty for drug insertion due to the disruption in the
chain packing, whereas the disordered fluid phase membrane has
a lower enthalpy barrier for drug insertion. This can be understood
more fully by considering that the gel lipid membrane consists
of ordered acyl chain trans conformers with a high degree of
chain coupling, whereas the fluid lipid membrane has high gauche
conformer content along with chain decoupling and rotation.[35,41]
Indeed, an increase in bilayer volume and especially bilayer area
occurs in fluid-phase membranes,[33,35] allowing greater room for
the drug to intercalate within the lipids.
Another observation from Fig. 8 is that the three TCAs show
similar partitioning behavior. The octanol/water partition coefficients of cationic amphiphilic drugs and TCAs have been shown to
have a positive correlation with their ability to disrupt both DPPG
and DPPC lipid membranes.[75] However, amitriptyline, which has
a much higher octanol/water partition coefficient than either protriptyline or nortriptyline, did not exhibit a significantly different
www.interscience.wiley.com/journal/jrs
effect on lipid Tm in DPPC or DPPG vesicles (Fig. 5). It should
be remembered that octanol/water partition coefficients are not
equivalent to liposome/water partition coefficients, especially for
amphiphilic molecules.[45,63] For example, liposome/water partition coefficients for protriptyline, amitriptyline, and nortriptyline
were determined using membranes composed of a mixture
of DPPG, DPPC, and cholesterol and found to be significantly
larger than the octanol/water partition coefficients (especially for
nortriptyline and protriptyline, see Fig. 1).[2] The octanol/water
partition coefficients were not able to predict the partitioning
behavior of the TCAs (Fig. 8). Amitriptyline has significantly higher
hydrophobicity (i.e. octanol/water partition coefficient) than either protriptyline or nortriptyline, so it might be expected to show
greater partitioning, at least in the neutral DPPC vesicles. Instead,
all three drugs appeared to have similar partitioning behavior in all
three lipid environments studied. This is expected on the basis of
the liposome/water partition coefficients of the three drugs listed
in Fig. 1. Complicating the partitioning discussion is a small proportion of neutral, deprotonated TCA at physiological pH, which
has been shown to have a higher DPPC liposome/water partition
coefficient than the charged form, especially when the TCAs have
an extra methyl group (such as with amitriptyline).[57] Protriptyline
has a higher pKa value (10.0) than either amitriptyline (9.4) or
nortriptyline (9.7),[41] so that if the unprotonated form was having
a large effect on the partitioning, then protriptyline would show
significantly less partitioning than the other two forms, which it
does not.
Confocal Raman microscopy and drug–membrane
interactions
There are several key advantages inherent in the characterization of
drug–membrane interactions by optical-trapping confocal Raman
microscopy. This technique overcomes the limited sensitivity of
Raman scattering through the use of an efficient, high-numericalaperture objective for excitation and scattered light collection in
a small sampling volume that is filled with the vesicle or vesicle
aggregate being studied. While the local concentrations of lipid
and partitioned drug are sufficiently high within this volume to
be readily detected, the concentration of drug averaged over
the entire sample can be quite small, close to in vivo levels. It is
estimated that typical plasma concentrations of TCAs would be
c 2009 John Wiley & Sons, Ltd.
Copyright J. Raman Spectrosc. 2010, 41, 498–507
Confocal Raman microscopy of tricyclic antidepressants in vesicle membranes
(a) 400
DPPC with 500 µM amitriptyline at 27 °C
DPPC with 500 µM amitriptyline at 46 °C
Raman Intensity / 10 PE
350
300
250
200
150
ring C=C stretch
1600 cm-1
100
50
0
1000
1500
2000
2500
3000
Wavenumber / cm-1
(b)
3.0
2.5
amitriptyline
2.0
I1600/I1730
+
N
1.5
CH3
CH3
1.0
0.5
0.0
25
30
35
40
45
50
with 5 nm bilayer thickness,[47] the total membrane volume is
2.2 µl. If the concentration of amitriptyline is 20 µM in solution,
there would be 32 mM drug in the membrane volume due to
partitioning. However, this would require 73 nmol of drug when
only 20 nmol are available in solution! Therefore, the results obtained from such an experiment would not represent the solution
concentration conditions which originally existed; i.e. there is not
an excess of 20 µM drug to partition into the lipid, so partitioning
lowers the actual concentration of drug in solution and in the lipid.
In contrast, a typical Raman microscopy experiment requires 40 µl
of 3 µM lipid dispersion, which gives a total membrane volume of
90 pl. If the concentration of amitriptyline is once again 20 µM, the
membrane volume would require 3 pmol of drug to reach equilibrium with the solution. Since there is 800 pmol of drug available
in solution, less than 0.4% depletion would take place. Therefore,
results from Raman microscopy truly represent the effects of a
20 µM amitriptyline solution. Thus, confocal Raman microscopy is
uniquely suited to investigate the influence of low concentrations
of lipophilic drugs on the phase transitions of vesicle membranes.
Despite the advantages of Raman microscopy, there are some
disadvantages. Relatively large liposomes (diameter >500 nm)
are required, in order for the vesicles to be visible through the eyepiece to guide them into the optical trap. While the electrostatic
repulsion in charged lipids makes them more likely to be unilamellar, phosphatidylcholine vesicles, especially of large diameter, can
be multilamellar,[83] and they appear to aggregate more as well.
The question of lamellarity might introduce data interpretation
problems if there is a very slow membrane permeability rate of
the drug. However, in the case of TCAs, it was shown that 30 min
of incubation at room temperature was more than enough time
to equilibrate the drug in both zwitterionic (i.e. multilamellar) and
anionic (unilamellar) lipid samples,[25,56] a condition which was
easily satisfied in the experiments presented here.
Temperature / °C
Figure 7. Tricyclic antidepressants exhibiting temperature-dependent
partitioning. (a) Comparison of the Raman spectrum of DPPC below and
above Tm in 500 µM amitriptyline solution. (b) Plot of the amitriptyline : lipid
ratio with increasing temperature in DPPC as revealed by the Raman
scattering peak ratio I1600 /I2847 .
J. Raman Spectrosc. 2010, 41, 498–507
Confocal Raman microscopy is shown to be an effective technique
to elucidate drug–membrane interactions using micromolar
concentrations of both lipid and drug. The results are more relevant
to the biological situation because low concentrations of drug
can be investigated while problems associated with depletion
of the drug in solution are avoided by working at even lower
lipid concentrations. Raman spectra provide conformational and
compositional information for the lipid and drug, respectively,
allowing membrane disordering and drug partitioning behavior
to be assessed. Raman spectra are collected from a ∼1.3 fl
volume of optically trapped lipid vesicles. In this study, it
has been shown that the cationic and amphiphilic TCA drugs
protriptyline, amitriptyline, and nortriptyline disorder and lower
the phase transition temperature of both zwitterionic and anionic
lipid membranes, while the effect on anionic membranes is
much greater, consistent with interactions between the anionic
lipid head groups and the cationic amphiphilic drugs. All three
drugs preferentially partition into anionic membranes and fluid
phase membranes, and overall partitioning results agree with
previously reported liposome/water partition coefficients but not
octanol/water partition coefficients.
Acknowledgements
This research was supported in part by the National Science
Foundation under Grant CHE-0654229 and by Eli Lilly and Co.
c 2009 John Wiley & Sons, Ltd.
Copyright www.interscience.wiley.com/journal/jrs
505
on the order of micromolars, with some of that concentration
bound to plasma proteins and thus unavailable for therapeutic
activity.[76,77] Moreover, amitriptyline causes hemolysis of red
blood cells and is toxic in a neurological animal model at ∼10
mM.[57,67,78] The critical micelle concentrations of TCAs are in the
low millimolar range,[79] above which TCAs act as detergents that
could dissolve lipid membranes.[44,79 – 82]
Biologically relevant (µM) drug concentrations cannot be
controlled in traditional methods to study lipid phase transitions
such as DSC and NMR, where high (mM) concentrations of lipid are
required to observe a response. Thus, if it is desired to examine
the membrane effects of a highly lipophilic drug at micromolar
concentrations in the presence of millimolar lipid, the drug in
solution would be quickly depleted, where there is not sufficient
drug available to partition into the membrane without depleting
the drug concentration in solution. For example, amitriptyline
has a liposome/water partition coefficient (logD) of 3.21.[44,63]
This means that there will be 1600-fold greater concentration
of amitriptyline in the lipid membrane than in solution. Thus,
in a typical DSC experiment that requires 1 ml of a 3 mM lipid
dispersion, with vesicles that are unilamellar and 1 µm in diameter
Conclusions
C. B. Fox and J. M. Harris
DPPC aboveTm
DPPC below Tm
2.0
DPPG above Tm
DPPG below Tm
DPPC:DPPG above Tm
DPPC:DPPG below Tm
1.5
I 1600 / I 1730
1.0
0.5
0.0
protriptyline
0
100
amitriptyline
200
300
400
500
0
100
nortriptyline
200
300
400
500
0
100
200
300
400
500
Drug concentration / µM
Figure 8. Partitioning of increasing concentrations of protriptyline (left), amitriptyline (middle), and nortriptyline (right) in DPPC, DPPG, and DPPC : DPPG
1 : 1 below and above Tm .
Additional support to Chris Fox from a University of Utah Graduate
Research Fellowship is gratefully acknowledged.
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