Interaction of piperidin derivative of mannich base with DPPC liposomes

Article abstract of DOI:10.1021/jp311825h

The long chain Mannich bases, especially with the piperidine and morpholine groups, display very promising antimicrobial activity. In order to extend our knowledge on their impact on biological systems, we examined the interactions of the 5-pentadecyl-2-((piperidin-1-yl)methyl)phenol (PPDP) with model lipid membrane by means of differential scanning calorimetry (DSC) and fluorescence measurements. The small unilamellar vesicles of dipalmitoylophosphatidylcholine (DPPC) with different piperidine Mannich base concentration were investigated as a function of the increase of temperature. The phase separation accompanied by the rise of the transition enthalpy of both subcomponents, the increase of the function of the GP values of Laurdan versus the wavelength of excitation in the gel phase of PPDP/DPPC systems, and no remarkable differences in the fluorescence anisotropy of PPDP molecules in lipid environment for different mixtures of PPDP/DPPC was observed. Additionally, it was shown that PPDP itself interdigitated in solid state.

Full text of DOI:10.1021/jp311825h

Article
pubs.acs.org/JPCB
Interaction of Piperidin Derivative of Mannich Base with DPPC
Liposomes
Katarzyna Cieslik-Boczula,*,† Rafał Michał Petrus,† Gottfried Kohler,‡ Tadeusz Lis,† and Aleksander Koll†
́
̈
†Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw, Poland
‡Max F. Perutz Laboratories, Department of Computational and Structural Biology, University of Vienna, Campus Vienna Biocenter
5/1, Vienna 1030, Austria
S
* Supporting Information
ABSTRACT: The long chain Mannich bases, especially with the piperidine and
morpholine groups, display very promising antimicrobial activity. In order to
extend our knowledge on their impact on biological systems, we examined the
interactions of the 5-pentadecyl-2-((piperidin-1-yl)methyl)phenol (PPDP) with
model lipid membrane by means of differential scanning calorimetry (DSC) and
fluorescence measurements. The small unilamellar vesicles of dipalmitoylophos-
phatidylcholine (DPPC) with different piperidine Mannich base concentration
were investigated as a function of the increase of temperature. The phase separation accompanied by the rise of the transition
enthalpy of both subcomponents, the increase of the function of the GP values of Laurdan versus the wavelength of excitation in
the gel phase of PPDP/DPPC systems, and no remarkable differences in the fluorescence anisotropy of PPDP molecules in lipid
environment for different mixtures of PPDP/DPPC was observed. Additionally, it was shown that PPDP itself interdigitated in
solid state.
direct contact with the Mannich compounds if they were
located in the membrane of the host cells. On the other hand,
there is a question if these molecules have any effect on the
membrane structure and other physicochemical properties, and
whether they can change the natural functions of cell
membranes. The largely flexible character of lipid aggregation
systems results from the low energetic barrier of rearrangement
of the lipid structures, strongly dependent on chemical
composition.10−16 It was shown that both PPDP and 3-
pentadecylphenol, as amphiphilic compounds, can easily build
into the lipid bilayer.17−19 In our previous studies, we stated
that the 3-pentadecylphenol changes significantly the lipid
phase transitions, induces phase separation, and forms a new
type of aggregates like micelles.17−19 The IR spectroscopic
studies of the DPPC membranes mixed with PPDP
demonstrated the influence of the piperidine Mannich base
mainly on the trans/gauche conformation of lipid hydrocarbon
chains.17 In this paper, we reveal further effects of PPDP on the
properties of the DPPC liposomes. In order to understand
better the thermo-structural behavior of water dispersion of the
DPPC liposomes mixed with different mol % of PPDP,
differential scanning calorimetry (DSC) was carried out. To
confirm the existence of the phase separation in the new
PPDP/DPPC membranes, fluorescence spectroscopy measure-
ments were performed, using Laurdan as a probe. A new type of
phases was proposed to be formed in the presence of the
piperidine Mannich base molecules.
1. INTRODUCTION
The Mannich bases, which have been reported as potential
biological agents, find an extraordinarily wide pharmaceutical
application as antitubercular,1 antimalarial,2 vasorelaxing,3
analgesic,4 and anticancer drugs.5 They exhibited activity
against Maurine P388 lymphocytic leukemia (Wi Dr Colon
cancer) in vitro.6 The long-chain derivatives play an important
role in the emulsification process.7 Moreover, a new trend in
the Mannich base actions appeared in their biocidal activity
toward various types of microorganisms. It was shown that the
long chain Mannich bases, especially with piperidine and
morpholine as the secondary amine moiety, exhibit a very high
biocidal activity toward Gram positive bacteria (Staph. Cocu.,
Bacillus), Gram negative bacteria (Salmonella, E. coli), fungi (A.
terrus, A. flavus), and yeast (Candida).8 In this paper, we have
studied 5-pentadecyl-2-((piperidin-1-yl)methyl)phenol
(PPDP), which is a new example of the long chain Mannich
base.
The biocidal activity of the Mannich bases depends mainly
on the chemical structure and their surface activity.9 The
antipatogenic action can be explained on the basis of the
superior ability of long tail Mannich bases to cross the lipid
membrane, mainly because of their lipophilic nature and
binding with the receptor sites. They anchor by its tail in the
hydrophobic core of the lipid bilayer, whereas a more polar
“head” is exposed to the surface of the membrane. The
presence of the Mannich bases in the cell membrane may cause
the effective resistance against the different types of micro-
organisms. The first step of many infections is the formation of
a direct contact between a pathogen and the plasma membrane
of host cells. Thus, the cells of bacteria and fungi could have
Received: December 1, 2012
Revised: February 15, 2013
Published: February 20, 2013
© 2013 American Chemical Society
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Figure 1. The structure and atom-numbering scheme for the PPDP molecule. The intramolecular O1H1···N8 contact, stabilizing the PPDP
structure, is shown as a dashed line.
of Laurdan were performed with a Perkin-Elmer LS 50-B
spectrofluorimeter, equipped with a thermostatted cell holder.
For temperature-dependent fluorescence intensity measure-
ments, the excitation wavelength was 390 nm. The emission
spectra were acquired over the range 400−560 nm. The
excitation wavelength was 320−400 nm for the fluorescence
excitation wavelength dependence studies. The Laurdan
generalized polarization (GP) was calculated using the
following equation adapted from the work of Parasassi et
al.:24,25
2. MATERIALS AND METHODS
The dipalmitoylophosphatidylcholine (DPPC) was purchased
from Avanti Polar Lipids, Germany, with a purity of >99.8%.
The fluorescence probe Laurdan (2-(dimethylamino)-6-dodec-
anoyl-naphthalene) was purchased from Molecular Probes
(Eugenie, OR) and the 3-pentadecylphenol (PDP) was
obtained from Sigma-Aldrich, Germany, both with a purity of
>95%. All compounds were used without further purification
procedure. The 5-pentadecyl-2-((piperidin-1-yl)methyl)phenol
(PPDP) was a product of Mannich condensation of 3-
pentadecylphenol, formaldehyde, and secondary amine. The
synthesis was performed according to refs 20 and 21. After
crystallization from methanol/water (1:1) solution, the next
step for checking and/or further purification of the product was
the TLC procedure on silica gel G type 60 with a solvent
composition of EtOAc/light petroleum/NH4OH (50:50:7).
The rest of the chemicals used in the experiments were of
analytical grade.
2.1. X-ray Structure Analysis. The X-ray data were
collected at 100 K using an Xcalibur PX diffractometer (ω and
φ scan technique).22 The experimental details and the crystal
data are given in Table S1 (Supporting Information). The
structure was solved by direct methods and refined by full-
matrix least-squares on F2 using the SHELXTL package.23 All
non-hydrogen atoms were refined with anisotropic thermal
parameters. All hydrogen atoms were positioned geometrically
and added to the structure factor calculations but were not
refined. Crystallographic data for the structural analyses
reported in this paper have also been deposited with the
Cambridge Crystallographic Data Centre (CCDC), number
903461. Copies of the information may be obtained free of
charge from the Director, CCDC, 12 Union Road, Cambridge
CB2 1EZ, U.K. (fax: +44-1223-336033; e-mail: deposit@ccdc.
cam.ac.uk; home page: http://www.ccdc.cam.ac.uk).
GP = (I440 − I490)/(I440 + I490
)
where I440 and I490 are the fluorescence emission intensities at
the blue and red edges of the emission spectrum, respectively.
Temperature-dependent intensity of the steady-state fluo-
rescence emission and anisotropy of PPDP molecules in water
dispersion of the DPPC liposomes was obtained using a PiStar-
180 spectrometer (applied Photophysiscs, U.K.) equipped with
a Peltier temperature controller. The excitation wavelength was
generally set to 280 nm.
2.4. DSC Measurements. The SUV liposomes with
different PPDP contents were prepared as described above.
The final PC lipid concentration was 5 mg/mL. The
calorimetric scans were recorded using the VP-DSC micro-
calorimeter. Individual samples were scanned six times for up
scans and six times for down scans. A scan rate was 90 and 60
°C/h for heating and cooling cycles, respectively. The prescan
and postscan thermostat was set at 15 and 10 min, respectively.
The samples were measured immediately after preparation. The
obtained endotherms were analyzed by using the Grams
software. The area under the transition profiles was used to
calculate the molar enthalpy change accompanying phase
transition (called further the transition enthalpy).
3. RESULTS
2.2. Preparation of Liposomes. Small unilamellar vesicles
(SUVs) with a varying PPDP/lipid composition have been
prepared. Mixed compounds from stock solutions in chloro-
form were dried under a stream of nitrogen. The residual
solvent was removed under a vacuum for 2 h. Dry lipid films
were hydrated by the addition of 1 mL of 10 mM phosphate
buffer, pH 7.2, during 10 of the cooling/heating processes.
Finally, liposomal suspensions were extruded through the filter
with 100 nm in the diameter of pores (LiposoFast with
polycarbonate filter, Avestin, Canada).
2.3. Fluorescence Measurements. The DPPC liposomes
mixed with 30 mol % PPDP compound were prepared as
described above. Unilamellar liposomes with a diameter of
about 100 nm were incubated with Laurdan fluorescence probe
in darkness for 30 min at room temperature. The final
concentration of Laurdan was 1.5 and 200 μM for
phospholipid. The steady state emission and excitation spectra
3.1. The Crystal Structure of PPDP Compound. The
asymmetric unit of PPDP contains one molecule, as shown in
Figure 1. The piperidine ring adopts a chair conformation, as
confirmed by the Cremer and Pople puckering parameters,26 or
the endocyclic torsion angles shown in Table 1. The angle with
the Cremer and Pople plane normal of the N8−C8 bond of
72.92(2)° affirmed an equatorial orientation of the 2-methyl-5-
pentadecylphenol groups attached to the heterocyclic ring. The
dihedral angle between the least-squares plane of the aromatic
ring and the pentadecyl part equals 5.7(2)°. The largest
deviations from planarity are observed for peripheral C13 and
C27 atoms of hydrophobic chain with values of 0.203(3) and
0.169(3) Å, respectively. In the PPDP molecule, there is a
strong intramolecular hydrogen bond of O−H···N type,
characteristic of the Mannich bases.27−29
The arrangement of the PPDP molecules in the crystal unit is
mainly determined by the weak C−H···C interactions. Such
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visible graphical representation of that head-to-head interaction
between neighboring PPDP molecules is shown in Figure 3B.
3.2. The Effect of the PPDP on the Phase Behavior of
DPPC Liposomes. The calorimetric behavior of the PPDP/
DPPC mixtures with an increasing mole concentration of the
Mannich base component is shown in Figure 4. For the pure
DPPC liposomes, the pretransition (Tp) and main phase
transition (Tm) were monitored at 34.5 and 41.5 °C,
respectively. Only in the lowest mole fraction of PPDP (up
to 10 mol %), the broad with a low intensity pretransition peak
was still observed. Additionally, in liposomes doped with 10
mol % of the compound under study, a small and flat deviation
from the baseline was also observed near the temperature 29
°C. The rise of the PPDP content in the DPPC membranes was
accompanied by the splitting of the main phase transition peak
into two main subprofiles fitted with the Lorentz function.
Phase separation into two main subcomponents begins to be
clearly visible in the mixture of 20 mol % PPDP (Figure 4).
With the increase of the PPDP/DPPC mole ratio, the
contribution of the lower temperature process becomes
gradually more prominent, which may be seen in the evolution
of the shape of the DSC thermograms in Figure 4. As the
concentration of the admixture was increased, the position of
the maximum of the low temperature peak was slightly shifted
to the higher temperature, from around 35 °C (20 mol %
PPDP) to 36 °C in the PPDP-rich membranes (Figure 5A).
The similar half-width of that peak around 1.6 °C was almost 2
times narrower than that for the corresponding high-temper-
ature profile. The second subcomponent was represented by a
high-temperature subpeak, and it had a similar maximum
position, centered at around 38.5 °C for all mixed systems with
the exception of liposomes with 10 mol % PPDP, where Tm was
approximately 1 °C higher (see Figure 5A). Additionally, the
half-height width of the high temperature DSC profile rose with
the increase of the mole fraction of the admixture, from around
2 °C for 10/90 mol % mole ratio of the Mannich base to the
DPPC molecules to more than 3.5 °C for 30/70 and 50/50 mol
% mixtures. The proportion of the value of transition enthalpy
(ΔH) for the PPDP/DPPC liposomes under scrutiny and of
the value of the DPPC transition enthalpy (ΔH0) changed as a
Table 1. Selected Geometric Parameters for PPDP Molecule
in Crystalline State
Cremer and Pople
puckering parameters (Å,
deg)
endocyclic torsion angle (deg)
−56.0(3)
Q
q2
q3
θ
0.569(3)
N8C8C9C10
0.020(3)
−0.568(3)
177.9(3)
160(7)
C8C9C10C11
C9C10C11C12
C10C11C12N8
C11C12N8C8
C12N8C8C9
53.5(3)
−53.0(3)
56.6(3)
−59.5(3)
58.2(3)
φ2
contacts, shorter than the sum of the van der Waals radii for the
H and C, are summarized in Table 2. As presented in Figure 2,
Table 2. Hydrogen Bond and Weak Contact Geometry (Å,
a
deg) in Crystal
DH···A
DH
H···A
D···A
DH···A
O1H1···N8
0.84
0.99
0.99
0.99
1.97
2.74
2.86
2.84
2.690(3)
3.601(4)
3.716(4)
3.812(4)
144
146
146
169
C7H7A···C2i
C13H13A···C5ii
C11H11A···C5iii
a
Symmetry codes: (i) x − 1, y, z; (ii) x + 1, y, z; (iii) −x + 1, −y, −z.
the weak contacts located in the area of the polar head of PPDP
link the adjacent molecules into infinite chains, realized by the
action a-axis translation, which leads to the formation of
intermolecular interdigitation of pentadecyl side chains. It is
noteworthy that intra- and interplanar distances between the
planes defined by neighboring pentadecyl chains are,
respectively, 3.66 and 3.94 Å. A similar arrangement of the
side chains has been previously observed for 6-n-pentadecyl-
2,4-dihydroxy-benzoic acid30 and 4-cyano-4′-n-dodecylbiphen-
yl,31 which accounts for the tendency to favor the formation of
the intermolecular interdigitation pattern in the solid state.
The large contributions to the formation of the interdigitated
layers are also weak contacts between the methyl-piperidine
groups and C atoms of aromatic rings; see Figure 3A. A clearly
Figure 2. Structural interdigitated motif created by the interpenetrated A and B layers arranged by weak C−H···C interactions.
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Figure 3. Packing motif created by head-to-head interaction between neighboring PPDP molecules effectuated by weak contacts leading to the
formation of an infinite chain.
sensitive to the polarity and mobility of its environment. The
characteristic fluorescence properties of Laurdan result from the
availability of the solvent molecules surrounding the fluorescent
naphthalene residue to form the dipole states and to reorientate
along the probe’s excited state dipole.25,25,32 As the
concentration and molecular dynamics of water molecules
located in lipid bilayers differ in gel and liquid-crystal states of
membranes, the Laurdan is widely used for monitoring phase
transitions, domain formation, and changes in the hydration,
polarity, fluidity, and order of lipid membranes.24,25,32 The 12-
carbon aliphatic tail anchors the naphthalene moiety at the level
of the phospholipid glycerol backbone. Few water molecules
present in this region of the lipid bilayer are responsible for
dipolar relaxation of probe molecules. In the gel phase, the
locally excited state gives the emission with the maximum value
in the blue region. In the liquid-crystalline condition, the
Figure 4. The DSC thermograms of DPPC lipids in the presence of
different mol % of PPDP. Inset: an example of thermogram (PPDP/
increase of hydration and mobility of the lipid membrane
results in the red-shifted emission that originated from the
formation of a charge transfer excited state, stabilized by the
water dipole reorientation process. Additionally, in the lipid
environment, in the ground state, there are Laurdan molecules
with and without partial charge separation, stabilized by
surrounding dipoles. Thus, the plot of the GP values versus
the excitation wavelength provides the quantitative information
on the proportion of the Laurdan molecules surrounded by the
gel or the fluid phase of phospholipids. The emission spectra of
Laurdan in the low- and high-temperature phase states of the
pure DPPC and PPDP/DPPC liposomes are shown in Figure
6A and B, respectively. The blue emission band in both types of
liposomes has a maximum value of approximately 440 nm and
shifts to the red part with the increase of temperature. In the
liquid crystalline phase of the pure DPPC bilayer, one band
DPPC, 30/70 mol %) deconvolution.
function of doped compound concentration. That is illustrated
in Figure 5B, in which the relative enthalpy (ΔH/ΔH0) is
plotted versus the mole concentration of PPDP. The relative
enthalpy for the high temperature component manifested a
biphasic behavior in the function of the PPDP content. This
value first decreases in a low range of mol % of the compound
under study, and then increases for the mixtures with more
than 20 mol % of the long chain phenol Mannich base. The
second, low-temperature component, as opposed to the first
one, was accompanied by a sharp and high growth of its
transition enthalpy, which proves that with the increase of
PPDP mol % the ΔH/ΔH0 rapidly increases.
3.3. Laurdan Fluorescence Studies. Laurdan (2-
dimethylamino-6-lauroylnaphthalene) is a fluorescence probe
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Figure 5. The evolution of the phase transition temperatures (A) and the relative enthalpy changes (ΔH/ΔH0) (B) for two subcomponents present
in DPPC liposomes mixed with different PPDP mol %. Errors are given as standard deviation values of six measurements.
one, characteristic for a fluid lipid phase. Figure 6C presents the
GP values for λex = 390 nm plotted as a function of temperature
for the pure and mixed DPPC liposomes. The sharp decrease of
GP marks the temperature of the main phase transition in the
DPPC bilayers, whose value is around 41.5 °C, the same as
obtained from the DSC measurement. The presence of the
compound under study in the lipid membrane induces more
gradual reduction of the GP values in the range of phase
transition temperature. Below the phase transition of the pure
DPPC membrane, the values of GP lie around 0.55, at the
temperature of main phase transition reaching almost zero, and
finally, in the fluid state, they adopt a position of about
−0.3.24,25,32 In the PPDP/DPPC mixture, in a lower temper-
ature range, GP slightly shifts to higher values as compared with
the pure DPPC membranes, and at the highest temperatures, it
assumes a value of −0.1, which significantly differs from the GP
of pure DPPC liposomes. The GP profiles of emission of
Laurdan in the function of the wavelength of excitation for the
pure and PPDP-mixed DPPC liposomes at different temper-
atures are presented in Figure 7. In the gel phase of the DPPC
membrane, the GP values constitute a straight line, whereas, for
liquid crystalline state, GP becomes a descending function of
λex. The slope of GP versus λex relationship is positive when
different phases coexist inside the system. Differently from the
pure DPPC, the ascending dependence of the GP versus λex
was present already at the temperatures before the phase
transition, which indicates the non-homogeneity of the PPDP-
doped DPPC membranes in the gel phase.
3.4. Photophysics of PPDP in DPPC Liposomes. The
PPDP compound has the aminomethyl-phenol moiety, which is
the source of its intrinsic fluorescence. Within one molecule,
there is a proton-donor OH group in the neighborhood of the
proton-acceptor center - nitrogen atom of the piperidine
moiety, which makes it possible to form an intermolecular
hydrogen bond in the “head” part of the PPDP molecule. The
strong O−H···N interaction was already proved by the
presence of a broad νOH band, centered at around 2800
cm−1 in the PPDP dry film exposed on the ZnSe crystal in the
IR-ATR experiments.17 Additionally, in the crystalline state of
Figure 6. The Laurdan emission band in the pure DPPC (A) and
PPDP/DPPC (30/70 mol %) (B) liposomes. The Laurdan generalized
polarization (GP) values as a function of the temperature for the pure
and PPDP-mixed DPPC vesicles. The excitation wavelength was 390
nm.
PPDP, the relatively short distance between O and N atoms,
equal to 2.689(3) Å, indicates the strong H-bond interaction.
Previous fluorescence studies performed on different derivatives
of the Mannich bases have indicated that the excited state
intramolecular proton transfer process is influenced by solvent
molecules and it is more pronounced in the polar solvents (ref
29 and articles cited therein). The dual fluorescence was
observed from the PPDP molecules incorporated into the fully
with the maximum at 490 nm is observed. However, in the
mixture of 30 mol % PPDP with 70 mol % DPPC, there is still
the blue emission band which coexists with the red emission
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Figure 7. Generalized polarization values for Laurdan in the pure DPPC liposomes (A) and with addition of 30 mol % PPDP as a function of
excitation wavelength at different temperatures.
Figure 8. Fluorescence emission spectra of PPDP molecules in DPPC liposomes in gel (A) and liquid crystal phase (B). The DPPC vesicles had the
10, 30, and 50 mol % Mannich base. The excitation wavelength was 280 nm.
Figure 9. (A) Temperature dependence of the fluorescence intensity for different concentrations of PPDP in DPPC: 10 (dashed line), 30 (dotted
line), and 50 mol % (solid line). An integrated (total) fluorescence intensity of PPDP at excitation wavelength λext = 280 nm was measured. (B) The
first derivative of the fluorescence intensity curves is shown in part A. (C) Temperature dependence of the fluorescence anisotropy for different
concentrations of PPDP in DPPC: 10 (dashed line), 30 (dotted line), and 50 mol % (solid line). (D) The first derivative of the fluorescence
anisotropy curves is shown in part C.
hydrated DPPC bilayers (see Figure 8). In general, the
fluorescence properties of PPDP in liposomes were similar to
other aminomethyl-phenols dissolved in polar solvents (like
water and alcohols), when the excited sate proton-transfer
reaction was present.29 In both the gel and liquid-crystal phases
of the DPPC bilayer, two fluorescence bands of PPDP coexist.
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The blue emission band centered at around 328 nm originates
from the neutral (molecular) form of long chain phenol
Mannich base. The second emission band, with the maximum
value at around 370 nm, is ascribed to the ionic, proton-
transferred form present in the excited state. Figure 9A and B
display, respectively, the temperature dependence of the PPDP
fluorescence intensity and of its first derivative for various
PPDP concentrations in DPPC liposomes. The quenching of
the intensity of the PPDP fluorescence was observed as a
function of increasing temperature. Additionally, a sharp
decrease of emission intensity was noticed at the approximate
temperature of transition of PPDP/DPPC liposomes. The
maximum of the peak of the first derivative allowed us to
determine the temperature of phase transition for all the
PPDP/DPPC water mixtures under study. The intensity of the
derivative profile decreased with the rise of the mol %
contribution of the Mannich base in liposomes. During the
heating process, the conformational fluidization is accompanied
by the increase of the hydration of lipid bilayer, especially at the
temperature of chain-melting lipid phase transition, when this
process has a dramatic course. Thus, the polarity of the
environment of PPDP in the DPPC membranes has an
influence on the photophysiscs of the compound under study.
For a lower mol % of PPDP, the proportion of the intensity of
the blue emission band to the red one was higher in the gel
state, in comparison to a more disordered and highly hydrated
liquid-crystalline phase (see Figure 8). When the concentration
of the Mannich base reached 30 or 50 mol %, this difference
faded away. Figure 9C,D presents the temperature dependence
of the phenol Mannich base fluorescence anisotropy data and
their first derivatives 10, 30, and 50 mol % PPDP in the DPPC
liposomes. The thermal evolution of the values of the
fluorescence anisotropy was independent of the PPDP
concentration in the lipid membrane. The gradual decrease of
anisotropy present in the outside of the region of phase
transition was broken at the temperature of phase transition.
The abrupt increase of the mobility of the phenol-2 piperidine
moiety marks the main phase transition of doped liposomes.
of the basic features of the occurrence of the interdigitation
phase in many different lipid-containing systems is the growth
of the transition enthalpy,36−38 the interdigitation can be one of
the possible processes to be considered in the system under
study. The growth of the relative enthalpy is more evident in
the second type, i.e., the PPDP-rich domains. It starts to be
present in 20 mol % PPDP−DPPC mixtures, when the second,
low-temperature thermogram arises. A sharper growth of the
relative enthalpy of the low-temperature component indicates a
higher contribution of the more promoted, PPDP-rich, newly
formed domains.
In a higher temperature range, over Tm, in the PPDP/DPPC
liposomes, the GP values are a descending function of
excitation wavelength, which is typical for the liquid crystalline
phase of the pure DPPC. However, the GP values are distinctly
higher than those for the pure DPPC liposomes in the fluid
state, which accounts for the lower hydration state and/or
lower fluidization of the liquid crystalline phase of the PPDP-
mixed lipid membranes. The comparison of the steady-state
fluorescence of the PPDP molecules in gel and liquid-crystal
phases for different mol % of the PPDP content in the DPPC
bilayer showed the decrease of the polarity of the systems under
study, with the rise of the concentration of the long chain
Mannich base which are less polar molecules than DPPC. The
contribution of the proton transfer reaction in the excited state,
which strongly depends on the polarity of the environment,29
decreases with the growth of PPDP mol % in lipid bilayers.
That feature is manifested by the lowering of the intensity of
the red emission band in Figure 8. After the chain-melting
phase transition, the polarity of the PPDP environment
increased evidently in the liquid-crystal state as compared
with the gel phase, only for mixtures with a lower PPDP
content. On the other hand, the main phase transition of the
DPPC bilayer, as is commonly known, is accompanied by the
increase of the fluidization and hydration of the membrane,
which causes the lower increase of population of the excited
state proton transfer form than in the DPPC membranes mixed
with 30 and 50 mol % PPDP. The similar evolution of the
fluorescence anisotropy of PPDP in the whole temperature
range for 10, 30, and 50 mol % PPDP suggests that the
admixture can change only the polarity of the lipid membrane
without noticeable alterations of the order parameter (see
Figure 9C,D). The measured values of the PPDP anisotropy
averaged the output signal over the whole liposomes, where the
phase separation is present. Therefore, it is difficult to state
clearly whether the emerging phases are not accompanied by
distinct changes of arrangement of the PPDP−DPPC
aggregates. It is commonly known from different studies of
structures of micelles or hexagonal phases that their formation
should be manifested by a considerable difference in anisotropy
values, as compared with the one for the pure DPPC bilayers.
On the other hand, the interdigitated state can be accompanied
by minor anisotropy changes.
4. DISCUSSION
The phase separation shown by the splitting of the DSC profile
of the PPDP/DPPC liposomes into two main subpeaks can
possibly mean that either the whole system undergoes two
separate transitions, one after another, or that there are two
separate regions (domains) with different chemical composi-
tions and/or different structures, and each of them is subject to
its own phase transition. The Laurdan fluorescence measure-
ments clearly showed the non-homogeneous gel phase in the
PPDP-mixed DPPC systems. The positive GP(λex) slope at the
temperatures below Tm indicates a mixture of different quasi-gel
phases present in separate domains. The simplest event that
could result in the domain formation would be the presence of
the PPDP-rich and PPDP-poor regions inside the membrane,
attributed to the incomplete miscibility of these two
components, analogous to cholesterol-phospholipid and to
many other systems.12,13,33−35 The PPDP-poor regions show
the biphasic evolution of the relative enthalpy (ΔH/ΔH0) as a
function of the PPDP content (see Figure 5). At a lower
Mannich base concentration, the relative enthalpy of the high-
temperature DSC peak decreases with the increase of the
concentration of doped compound. However, from 20 mol %
PPDP, the characteristic growth of the enthalpy indicates the
formation of a new phase in the PPDP/DPPC bilayers. As one
The crystallographic data showed the favorable three-
dimentional packing of the PPDP molecules. The geometry
of this compound allows tight packing in both polar and apolar
regions of PPDP aggregates. The PPDP molecules in the crystal
cell have the hydrocarbon chains in the long expanded trans
state. The close contacts of hydrophobic tails were allowed due
to the interdigitated process, which is clearly a consequence of
the larger energy gain from the strongest van der Waals
interactions. In the interdigitated state, the molecules from one
monolayer interpenetrate the free spaces between molecules of
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The Journal of Physical Chemistry B
Article
the opposing layer. That phenomenon can be observed in
different long-chain amphiphilic molecules, for example,
phospholipids. The DPPC bilayers can adopt the gel
interdigitated phase (LβI) in the presence of surface active
molecules or in higher pressure conditions.36,39−41 The
methanol, ethanol, or acetonitrile molecules create a small
void which can be entered by the hydrocarbon chains from
phospholipids in the opposing layer.36,39−41 The incorporation
of the PPDP molecules into the DPPC bilayer leads to the
distinct changes of the structure and physicochemical proper-
ties of lipid membranes, as shown in the above-presented
microcalorimetric and fluorescence studies. The PPDP
molecules have a conical shape. The relatively large volume
of the hydrophilic head, as compared with the narrow but long
tail, can induce the formation of energetically undesirable “free
volume” between the tails in the hydrophobic region of mixed
bilayers. Filling these gaps with the chains from the
hydrophobic region leads to the formation of new phases in
studied mixtures. Different types of structures of the PPDP/
DPPC aggregates can be expected. If free volumes are be
reduced when the free spaces between tails of one monolayer
are interpenetrated by the chains from the opposing one, then
the interdigitated phase can be formed. Additionally, at higher
mol % of the Mannich base, as a result of the nonuniform
distribution of the admixture, there are regions consisting
mainly of PPDP molecules, which can aggregate in a similar
manner as in the crystal state.
ACKNOWLEDGMENTS
■
This work was supported by the Foundation for Polish Science
- the programme POMOST, cofinanced by the European
Union within European Regional Development Fund (V
edition 2012) and by Grant No. N N204 150338 from the
̈
National Science Centre. We thank the OAD (Proj. PL-05/
2011) and Polish Ministry of Science and Higher Education
(grant-in-aid for scientific research and development for young
scientists, 2012) for additional financial support.
ABBREVIATIONS USED
■
DPPC - 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; PPDP -
5-pentadecyl-2-((piperidin-1-yl)methyl)phenol; DSC - differ-
ential scanning calorimetry; SUV - small unilamellar vesicles
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