Porphyrin-Based Probe for Simultaneous Detection of Interface Acidity and Polarity during Lipid-Phase Transition of Vesicles

Article abstract of DOI:10.1021/acs.langmuir.9b02781

Biochemical activities at a membrane interface are affected by local pH/polarity related to membrane lipid properties including lipid dynamics. pH and polarity at the interface are two highly interdependent parameters, depending on various locations from the water-exposed outer surface to the less polar inner surface. The optical response of common pH or polarity probes is affected by both the local pH and polarity; therefore, estimation of these values using two separate probes localized at different interface depths can be erroneous. To estimate interface pH and polarity at an identical interface depth, we synthesized a glucose-pendant porphyrin (GPP) molecule for simultaneous pH and polarity detection by a single optical probe. pH-induced protonation equilibrium and polarity-dependent π-πstacking aggregation for GPP are exploited to measure pH and polarity changes at the 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG) membrane interface during DMPG phase transition. An NMR study confirmed that GPP is located at the interface Stern layer of DMPG large unilamellar vesicle (LUV). Using UV-vis absorption studies with an adapted analysis protocol, we estimated interface pH, or its deviation from the bulk phase value (ΔpH), and the interface polarity simultaneously using the same spectra for sodium dodecyl sulfate micelle and DMPG LUV. During temperature-dependent gel to liquid-crystalline phase transition of DMPG, there was ～0.5 unit increase in ΔpH from approximately -0.6 to -1.1, with a small increase in the interface dielectric constant from ～60 to 63. A series of spectroscopic data indicate the utility of GPP for evaluation of local pH/polarity change during lipid phase transition of vesicles.

Full text of DOI:10.1021/acs.langmuir.9b02781

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New Concepts at the Interface: Novel Viewpoints and Interpretations, Theory and
Computations
A porphyrin-based probe for simultaneous detection of interface
acidity and polarity during lipid phase transition of vesicle
Rini Majumder, Snigdha Roy, Kentaro Okamoto, Satoshi Nagao, Takashi Matsuo, and Partha Pratim Parui
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b02781 • Publication Date (Web): 10 Dec 2019
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Langmuir
A porphyrin-based probe for simultaneous detection of interface
acidity and polarity during lipid phase transition of vesicle
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Rini Majumder,† Snigdha Roy,† Kentaro Okamoto,‡ Satoshi Nagao,‡ Takashi Matsuo,*‡ and Partha
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Pratim Parui*†
†Department of Chemistry, Jadavpur University, Kolkata 700032, India
‡Division of Materials Science, Graduate School of Science and Technology, Nara Institute of Science and Technology
(NAIST), 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
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ABSTRACT: Biochemical activities at a membrane interface are affected by local pH/polarity related to membrane lipid properties
including lipid dynamics. pH and polarity at the interface are two highly interdependent parameters depending on various locations
from the water-exposed outer-surface to the less-polar inner-surface. The optical response of common pH or polarity probes are
affected by both the local pH and polarity; therefore, estimation of these values using two separate probes localized at different
interface depths can be erroneous. To estimate interface pH and polarity at an identical interface depth, we synthesized a glucose-
pendant porphyrin (GPP) molecule for simultaneous pH and polarity detection by a single optical probe. pH-induced protonation
equilibrium and polarity-dependent -stacking aggregation for GPP are exploited to measure pH and polarity changes at the 1,2-
dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) membrane interface during DMPG phase transition. An NMR study
confirmed that GPP is located at the interface Stern layer of DMPG large unilamellar vesicle (LUV). Using UV-vis absorption studies
with an adapted analysis protocol, we estimated interface pH, or its deviation from the bulk phase value (pH), and the interface
polarity simultaneously using the same spectra for sodium dodecyl sulfate (SDS) micelle and DMPG LUV. During temperature
dependent gel to liquid-crystalline phase transition of DMPG, there was ~0.5 unit increase in pH from approximately 0.6 to 1.1,
with a small increase in interface dielectric constant from ~60 to 63. A series of spectroscopic data indicate the utility of GPP for
evaluation of local pH/polarity change during lipid phase transition of vesicles.
at an identical interface depth using a single optical probe is pro-
posed here to evaluate either of these two parameters. This is
particularly useful for temperature-dependent studies because
polarity is intrinsically related to temperature.
INTRODUCTION
Cellular events at phospholipid membrane interfaces are asso-
ciated with the structural dynamics of the membrane, e.g., the
lipid phase transition from tightly-packed gel to flexible liquid-
crystalline state at room temperature.1, 2 Because the membrane
becomes softer in the liquid-crystalline state, endocytosis and
exocytosis reactivity are accelerated above the lipid melting
Porphyrin derivatives have characteristic absorption bands
around 400 nm (Soret band) and 550 nm (Q band). A large ex-
tinction coefficient (~104–105 M–1cm–1) at the Soret band is use-
ful as an indicator reagent.12 Porphyrin derivatives work as pH
indicators because of the significant UV-vis spectral changes
during protonation at inner nitrogen atoms in the ring. For ex-
ample, Liu and co-workers reported that tetraphenylporphyrin
(TPP)-type compound 1 (Chart 1) works as a pH indicator in the
range of pH 4–5, where the electron-donating phenoxy moiety
is a key structural factor of pH detection in this range.13 Further-
more, deprotonated porphyrin compounds (i.e. non-protonated
state at the inner nitrogen atoms) readily form molecular aggre-
gation states (known as J-aggregation or H-aggregation)
through intermolecular hydrophobic -stacking under polar en-
vironments, whereas the porphyrin compounds exist as a mon-
omer state in non-polar solvents.14, 15 The aggregation behavior
is also reflected in the shape of UV-vis spectra of porphyrins.
Therefore, the UV-vis spectra of porphyrin derivatives reflect
both environmental pH and polarity. According to these charac-
teristics, we expected that TPP-type porphyrins are suitable for
simultaneously elucidating the local pH and polarity at a mem-
brane interface.
4
temperature.3, Mechanical signal propagation phenomenon
such as nerve pulses transmittance is strongly associated with
lipids phase transition.5 However, several other independent in-
vestigations have revealed that those membrane biochemical ac-
tivities are highly sensitive to local pH and polarity surrounding
the membrane.6-8 In this context, we believe that any changes in
these physicochemical properties at the membrane interface
during lipids phase transition may have profound roles to affect
aforementioned membrane reactivity.
Self-assembly occurs at the interface whereby charged/un-
charged polar headgroups separates from nonpolar (lipid/sur-
factant acryl chain) phase and assemble at the polar (aqueous)
phases. Thus, pH and polarity at the interface are different from
that of the bulk medium. We have recently developed a new in-
terface pH and polarity monitoring method for micelles and ves-
icles using two separate pH and polarity sensitive chromophore
probes that interact with the interface.9, 10 However, interface pH
and polarity are highly interrelated physicochemical parameters
along various interface locations. For example, a decrease of
polarity with increasing interface depth towards the hydropho-
bic phase may decrease interface acidity because of a lack of
H3O+ conduction ability11. pH-induced optical changes for a pH
probe are frequently affected by solvent polarity9, 10 and vice
versa. Thus, the precise measurement of pH and polarity using
two separate probes localized at different interface depths can
be problematic. The simultaneous detection of pH and polarity
In this study, we demonstrate the utility of moderately wa-
ter-soluble glucose-pendant porphyrin GPP (2) for monitoring
pH and polarity simultaneously at the membrane interface. GPP
(2) adopts both a neutrally charged (basic form) and cationic
forms (acidic form) depending upon the surrounding pH. This
assures: 1) binding of GPP (2) to the Stern layer of the anionic
interface assisted by the cationic charge in the acidic-form por-
phyrin unit and/or by the dual characteristics of GPP (2) with
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the polar glucose residues and the nonpolar basic-form porphy- were filtered through Millipore membrane filter (0.22 µm) be-
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rin unit; and 2) polarity-induced aggregation behavior of GPP
(2) without affecting the intrinsic protonation property at the
porphyrin core. Firstly, we demonstrate the protocol for syn-
chronized evaluation of pH and polarity in homogeneous sol-
vents. Next, we applied this protocol for the simultaneous eval-
uation of local pH and polarity at the interface of self-assembled
sodium dodecyl sulfate (SDS) (3) micelles and 1,2-dimyristoyl-
sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DMPG)
(4) vesicles. In addition, we succeeded in the quantitative meas-
urement of interface pH and polarity changes during tempera-
ture-induced DMPG phase transition in vesicles.
fore spectroscopic measurements. For temperature variation
measurement, the measuring solutions were equilibrated at the
particular temperature for 5 min. All measurements were carried
out at least three times to check the reproducibility. Different
buffer compositions were used to attain a particular medium pH:
sodium citrate/sodium phosphate for pH 3.05.0; sodium caco-
dylate-HCl for pH 5.06.0; HEPES-NaOH for pH 6.07.0. The
required amount of GPP (2) was added to a buffer solution in
the presence and absence of amphiphilic systems, with an addi-
tion of either 0.1 M NaOH or 0.1 M HCl to adjust the desired
pH, if required. The dielectric constants of mixed solvent me-
dium were determined according to a previous report.17
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EXPERIMENTAL SECTION
Chart 1. Chemical structures of TPP-type porphyrins
and lipid surfactants.
Materials and Instruments. Porphyrin 1 was prepared accord-
ing to a previously reported method.13 GPP (2) and its related
intermediate species were synthesized as described in Support-
ing Information. SDS (3) and DMPG (4) were purchased from
Sigma Aldrich Chemicals (USA) and used without further puri-
fication. DMPG large unilamellar vesicle (LUV) was prepared
as described below. For preparation of buffers and other analyt-
ical measurements, milli-Q Millipore® 18.2 MΩ.cm water was
used. Medium pH was measured with a Systronics digital pH
meter (Model No. 335). Other chemicals were obtained from
conventional vendors. UV-vis spectral changes were followed
using a Shimadzu UV-2550 double beam spectrophotometer
with a thermostated cell holder. The average particle size for
lipid vesicles were evaluated by dynamic light scattering (DLS)
measurement with Malvern Instruments, DLS-nano ZS,
Zetasizer, Nanoseries. Cryo-transmitted electron microscopy
(TEM) experiments were performed with an FEI Tecnai F20
electron microscope equipped with Gatan K2 summit direct de-
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tection device. H NMR spectra were collected using a JEOL
NM-ECA600 or JNM-ECX400 spectrometers. Differential
scanning calorimetry (DSC) measurements were carried out us-
ing a Malvern MicroCal VP-DSC calorimeter. Electron ioniza-
tion mass (EI-MS) measurements were conducted using a JEOL
JMS-700 mass spectrophotometer. Matrix-assisted Laser De-
sorption Ionization mass (MALDI-TOF-MS) spectra were
measured using a JEOL JMS-S3000 mass spectrophotometer,
where trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenyli-
dene]malononitrile (DCTB) was used as a matrix reagent.
RESULTS AND DISCUSSION
Synthesis of glucose-pendant porphyrin (GPP) 2. The syn-
thetic route of GPP (2) is shown in Scheme 1. The detailed syn-
thetic procedure is described in Supporting Information.
Scheme 1. Synthetic route of GPP (2).
Preparation of DMPG large unilamellar vesicle (LUV).9,16
DMPG was dissolved in 1.0 mL chloroform/methanol (5:1)
mixed solvent in 5 mL round bottom flask. The organic solvents
were removed rotary evaporator at 35 °C to prepare thin lipid
film. Any residual amount of organic solvent was completely
removed in vacuo for 3 h. For hydration of prepared thin film,
appropriate buffer solution at desired pH was added at 45 °C.
The solution was vortexed for 2.0 min for complete dissolution
of the lipids to form multi-lamellar vesicles. Seven cycles of
freeze-and-thaw were performed between 196 °C and 50 °C to
produce giant multilamellar vesicles. To obtain unilamellar ves-
icles (LUVs) of diameter ~100 nm, the liposome dispersion was
extruded 15-times through two stacked polycarbonate mem-
brane filters (Whatman) of 100 nm pore sizes equipped in a
Mini-Extruder system (Avanti Polar Lipid, USA). The temper-
ature throughout the LUVs preparation process before and after
lipid film hydration was maintained above 30 C.
UV-vis absorption studies. A Quartz cell with 1 cm path-
length was used for absorption measurements. All solutions
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Langmuir
A popular method for the synthesis of compound 8, a key
With an increasing amount of acetone in the buffer, a sharp ab-
sorption band around 420 nm appeared with concomitant deple-
tion of those broad bands (Figure 1A). The absorption band
around 420 nm is a typical feature of TPP-type porphyrins ob-
served in organic solvents.20 Although the absorbance changes
were not systematic up to 10% (w/w) of acetone with dielectric
constant () ~73 (due to change in aggregation fashion14, 21 (Fig-
ure S1)), the absorbance increased from ~0.17 to 0.44 upon the
addition of acetone from ~16% to 60% (w/w) with maintaining
isosbestic points at ~407 and 430 nm (Figure 1A). The quantity
of intensity changes at ~420 nm were very similar in other or-
ganic solvents than acetone (e.g. methanol and ethanol, protic
solvents) (Figure 1A and S2); suggesting that the observed UV-
vis spectral change is interpretable as not specific solvent effect
but as polarity effect in the range of solvent  from approxi-
mately 41 to 69. The series of observed spectral changes indi-
cate that the deprotonated GPP undergoes an interconversion
equilibrium between the aggregated form (denoted as dA-GPP)
and the monomer form (dM-GPP), where “d”, “A” and “M”
stand for “deprotonated”, “aggregated”, and “monomer”, re-
spectively.
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building block in the synthesis of GPP(2), is reflux of a propi-
onic acid solution of 4-hydoxybenzaldehyde (5) and pyrrole13
(the so-called Adler-Longo method18). However, the hydro-
philic property of 8 (insoluble in CH2Cl2 or CHCl3) caused dif-
ficulty in separation from turbid and gummy by-products. The
low solubility of 5 in CH2Cl2 prevented Lindsay porphyrin syn-
thesis19 using a Lewis acid catalyst. In contrast, the synthetic
route shown in Scheme 1 proved that column purification is not
required for obtaining porphyrin 8. To attach sugar moieties to
the periphery of the porphyrin ring, peracetylated glucose with
a chloroethyl linker (10) was reacted with the phenolic moiety
of 8 by Williamson synthesis to yield compound 11. The in-site
replacement of the chlorine atom in 10 with iodine using potas-
sium iodide (KI) was essential to increase the yield of 11. Fi-
nally, the acetyl groups in 11 were removed under basic condi-
tions to obtain GPP (2).
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GPP monomer/aggregate equilibrium: Estimation of po-
larity. Firstly, we attempted to evaluate medium polarity (die-
lectric constant ()) by the observation of UV-vis spectral
changes in buffer/acetone mixed medium (Figure 1).
In contrast to GPP (2), porphyrin 1 showed a distinct ab-
sorption band at ~420 nm in aqueous medium with comparable
intensity to that observed for GPP (2) in the presence of organic
solvents (Figure S3). The absorption pattern and intensity in-
creased a little (< 10%) in the presence of methanol or acetone
in buffer at pH 7.0. These results demonstrate that the UV-vis
spectra of porphyrin 1 are less sensitive on solvent polarity com-
pared to GPP (2). The spectral changes indicate that porphyrin
1 predominantly exists as a monomer form in the buffer solution
because of cationic ammonium moieties. Therefore, GPP (2) is
superior to 1 as a polarity indicator.
The dA-GPP to dM-GPP interconversion equilibrium was
found to depend on medium temperature as well. For the mixed
medium containing different acetone ratios (2450%) in buffer,
the intensity at ~420 nm for GPP (2; 2.5 M) increased gradu-
ally with rising temperature from 20 C to 45 C (Figure S4).
Both the increase in temperature and in acetone ratio displayed
a similar decrease in solvent  ~5.56.0 units (Figure 1A and
S4A).17 Namely, the temperature-induced absorbance change at
~420 nm is regarded as the temperature affected change in me-
dium .17 Therefore,  of GPP (2) localized environment can be
estimated by monitoring dA-GPP to dM-GPP interconversion
based on the absorbance change at ~420 nm. Accordingly, we
empirically correlate the absorbance change at ~420 nm and
medium . The negative logarithmic value for the amount of
intensity decrease at ~420 nm from the saturated intensity at 
< 35 under complete conversion to dM-GPP exhibited a linear
correlation with solvent  in the range from  ~40 to 70 (Figure
1B), and thus  can be estimated by using the eq.(1):
Figure 1. (A) UV-vis absorption spectra of GPP (2; 2.5 M) in 10.0 mM
sodium citrate/sodium phosphate buffer containing various amounts of ac-
etone% (w/w), pH 6.0 at 25 °C: red, 60% (41.5); violet, 51% (47.7);
dark cyan, 42% (53.4); purple, 33% (59.2); orange, 24% (64.5);
light green, 20% (67.0); blue, 16% (69.5) and gray, 73% (72.9).
The spectra in pure buffer (78.5) and acetone (21.0) medium are
shown by dark blue and black, respectively. The change in dielectric con-
stant () is depicted by the arrow. (B) The negative logarithmic values of
intensity difference at 420 nm from pure acetone with  ~21 (A21420) of other
various acetone/buffer mixed mediums with  ~41.5–69.5 (Ax420) are plot-
ted against . The values of dielectric constants were quoted from a previous
paper.17
log(A42021  A420x) = 0.13   + 10.3 (40 ≤  ≤ 70) (1)
21
x
where A420 and A420 represent the absorbance value at ~420
nm in acetone medium ( = 21) and the observed absorbance
for a solution at unknown polarity (i.e. an observation target),
respectively. The intercept and slope values are represented by
10.3 and 0.13, respectively.
In buffer (pH > 6.0) at 25 C, GPP (2, 2.5 µM) exhibited
broad absorbances consisting of weak overlapping intensities
spreading from ~400 to 440 nm (Figure 1A). This spectrum
shape is often observed for H-type aggregated porphyrins with
a small contribution of J-type aggregation in aqueous media,14
where the inner nitrogen atoms in GPP (2) are deprotonated.
GPP acid/base equilibrium: Estimation of pH. Next, we at-
tempted to develop the protocol for evaluation of pH at various
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polarity conditions based on the UV-vis spectra of GPP (2). In
Page 4 of 11
Electrostatic repulsion between positively-charged porphy-
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pH-dependent UV-vis absorption studies for GPP (2.5 µM) in a
buffer solution, a new absorption band at ~446 nm appeared
with the decrease in the broad band of dA-GPP on decreasing
pH from 5.5 (Figure 2A). The spectral change is due to the pro-
tonation at inner nitrogen atoms in the porphyrin ring. The ab-
sorbance increase saturated at pH below 3.5, suggesting a com-
plete protonation at inner nitrogen atoms in GPP.12 A much
sharper Soret band of the protonated GPP compared to dA-GPP
(Figure 2A) indicates that the protonated GPP exists in the mon-
omeric state. Namely, the protonated GPP is denoted as “pM-
GPP”, where “p” stands for “protonated” form. In the presence
of buffer containing different acetone ratios, the single isosbes-
tic point in the pH-dependent spectral changes indicate the ap-
parent one-step interconversion of two deprotonated forms (dA-
GPP and dM-GPP) (at high pH, abs ~ 420 nm) into protonated
pM-GPP (at low pH, abs ~ 450 nm and 685 nm) (Figure 2B and
S5). The whole porphyrin interconversion reflected in Figures
2A and 2B are summarized in Scheme 2.
rins with protonated inner nitrogen atoms prevents the hydro-
phobic -stacking interaction resulting in the existence of the
monomeric state, even in pure aqueous buffer. By following a
similar trend, another relatively weak absorption intensity at
~685 nm also increased systematically with decreasing pH, sug-
gesting that both intensities at ~446 and ~685 nm are associated
with the pM-GPP form (Figure 2A). Therefore, the equilibrium
mole-ratio of pM-GPP (XpM-GPP) can be determined by analyzing
the absorption intensities at ~446 nm and ~685 nm as follows:
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6.0
XpM-GPP = (ApH – A6.0)/(A3.5 – A
)
(2)
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where ApH and A3.5 represent the pH-dependent absorbance
and the saturated intensities below pH 3.5 at wavelength ()
~446 or ~685 nm, respectively. A6.0 denotes the absorption in-
tensity at ~446 nm or ~685 nm when GPP exists as deprotonated
state (dM-GPP and/or dA-GPP) at pH above 6.0. The mole-ratios
of pM-GPP (XpM-GPP) were plotted with pH (Figure 2C), and the
acid/base pKa for GPP was estimated to be ~4.2 by fitting data
points with a sigmoidal-Boltzmann equation (the data of first
raw in Table S1). The unknown pH of an aqueous medium can
be estimated from the pH vs. pM-GPP mole-ratio calibration
curve.
The pH vs. pM-GPP mole-ratio calibration curve in ace-
tone/buffer solutions shifted to the direction of lower pH com-
pared to the pure buffer solution (cf. black and gray lines in Fig-
ure 2C). The deviation reflects the polarity effect on the proto-
nation/deprotonation equilibrium. This indicates that the contri-
butions of solvent  and pH should be separated in the observed
UV-vis spectral changes in order to develop the protocol to es-
timate pH under variable polarity conditions.
Accordingly, we performed the pH-metric titration of GPP
under various polarity conditions to monitor the interconversion
from dA-GPP and/or dM-GPP to pM-GPP (Figures S5 (AE)).
Similar to the spectral change observed in buffer solutions, a
sharp band around ~450 nm for pM-GPP form gradually ap-
peared with decreasing pH from 6.0 to 2.5. The apparent
acid/base pKa decreased from ~4.10 to 3.35 with decreasing sol-
vent  from ~62.0 to ~42.0 at 25 C (Table S1, Figure S5). The
lack in solvation of cationic pM-GPP at low  results in the de-
crease in apparent pKa. Since the magnitude of A6.0 (in eq. 2) is
independent of medium polarity (  70), the pH dependent pM-
GPP/GPP ratio (XpM-GPP) can be estimated independently with-
out knowing the  of mixed solvents.
Figure 2. pH-dependent UV-vis analyses of GPP (2; 2.5 M) at 25 C; (A)
Spectral changes observed in 10 mM sodium citrate/sodium phosphate
buffer, pH 3.40, 3.70, 3.90, 4.05, 4.20, 4.30, 4.40, 4.55, 4.90, and 5.20; (B)
Spectral changes observed in the buffer containing 31%(w/w) acetone, pH
3.15, 3.40, 3.60, 3.90, 4.05, 4.20, 4.30, 4.40, 4.60, 4.90, 5.25, and 5.7. The
change in intensities with decreasing pH are shown in arrows. (C) pM-GPP
mole-ratio (XpM-GPP) analyzed from the intensity at ~450 nm (circle, solid
lines) and ~685 nm (triangle, broken lines) are plotted against pH: black, 10
mM sodium citrate/sodium phosphate buffer; gray, 31% (w/w) acetone con-
taining buffer.
For the acetone/buffer mixed medium, temperature depend-
ent pKa values are estimated and listed in Table S1. Interest-
ingly, a certain decrease in solvent  by either an increase in
acetone ratio or increase in temperature in the buffer/acetone
mixed medium exhibited a similar extent in acid/base pKa shift
(Table S1, Figures S5 and S6). These results suggest that the
temperature-dependent pKa shift for GPP is mostly associated
with the variation of solvent  (Figures S5 and S6). Therefore,
maintaining an identical temperature condition of the measuring
and calibrating solutions is a prerequisite to determine the GPP
environmental pH by utilizing pH-metric titration curves.
Scheme 2. pH and dielectric constant () dependent intercon-
version equilibrium among aggregated-deprotonated (dA-
GPP), monomeric-deprotonated (dM-GPP) and monomeric-
protonated (pM-GPP) forms of GPP.
Simultaneous estimation of pH and polarity in homogene-
ous solutions. Under complete protonation of GPP, the identi-
cal absorption intensity at various solvent  (Figure 2 for buffer
solutions, Figure S5 for acetone-containing buffer solutions,
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The protons signals at 7.5 ppm, 8.3 ppm and 9.1 ppm of 2 dis-
and in Figure S6 for solutions at different temperatures) indi-
1
2
3
4
5
6
7
8
played correlation signals with proton peaks at 4.1 ppm and 4.5
ppm, corresponding to the protons at the head moiety of DMPG.
Similar cross peaks were also observed in the measurement at
20 C (Figure S9). The results of 2D NMR spectral analysis
strongly indicate that GPP (2) locates on the interface Stern
layer of DMPG LUV regardless of temperature change. There-
fore, GPP (2) is suitable to measure pH and polarity at a similar
Stern layer location for DMPG LUV during the temperature-
induced lipid phase transition.
cates molar extinction coefficients () at all wavelengths does
not depend upon medium . However, when GPP becomes par-
tially protonated, the intensity change at ~420 nm is to be af-
fected not only by the -induced change in dA-GPP to dM-GPP
ratio for the deprotonated GPP, but also their protonation to
form pM-GPP. By evaluating XpM-GPP from eq. (2), the intensity
due to pM-GPP form at ~420 nm is calculated as Ap420  XpM-GPP
,
where Ap denotes the intensity for the fully protonated GPP
420
(XpM-GPP = 1). Therefore, the actual intensity of deprotonated
GPP (dA-GPP and/or dM-GPP) at ~420 nm can be obtained by
subtracting Ap420  XpM-GPP from the observed intensity (A420obs).
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Furthermore, (A420obs  Ap  XpM-GPP) was normalized by di-
420
viding it with the mole-ratio of deprotonated GPP (1  XpM-GPP
to use eq. (1) for the estimation of :
)
A420N = A420x = (A420obs  Ap420  XpM-GPP) / (1XpM-GPP
)
(3)
log(A42021  A420
)
x
= log[A42021  (A420obs  Ap420  XpM-GPP) / (1  XpM-GPP)]
= 0.13   + 10.3 (4)
Using eq. 4, an unknown  can be estimated by evaluating XpM-
and A420N from the intensities at ~450 or 685 nm and 420
GPP
nm, respectively. Notably, the more general eq. 4 than eq. 1 can
be used to estimate  under the conditions of various extent of
GPP protonation in a wide range of bulk pHs. Once the medium
 is known, pH of the medium can also be simultaneously esti-
mated from the same absorption spectrum by correlating XpM-
with the pH-metric calibration curve obtained under same
GPP
medium .
A series of test of the above procedure above demonstrated
that GPP (2) is a highly effective for simultaneous detection of
environmental pH and  using a single absorption spectrum.
Interaction of GPP with self-assembly interfaces. Encour-
aged by the proved utility of GPP (2) for simultaneous determi-
nation of the local polarity and pH around the molecule, we in-
vestigated the availability of GPP (2) for probing the local en-
vironment at the interface of lipid emulsions. Firstly, we at-
tempted to elucidate interaction fashions between GPP (2) and
1
DMPG LUV by 1H NMR spectroscopy. The full 1D H NMR
spectrum of the mixture of GPP (2) and DMPG LUV observed
at 40 C is shown in Figure 3A. Figure 3B shows the tempera-
ture dependency on 1D 1H NMR spectrum in the range from 7.0
to 10.0 ppm. The diameter of DMPG LUV at ~100 nm was con-
trolled by the lipid extrusion (see Experimental Section) and the
size distribution and morphology were further confirmed by
DLS and cryo-TEM analysis, respectively (Figures S7 and S8).
Figure 3. 600 MHz-1H NMR spectroscopic analysis for the mixture of
GPP(2) and DMPG LUV; (A) Whole 1D NMR spectrum at 40 C; (B) 1D
NMR spectra at various temperatures in the range from 7.0 ppm to 10.0
ppm; (C) NOESY spectrum at 40 C; Conditions: [2] = 1 mM, [DMPG LUV
(d ~100 nm)] = 8 mM in D2O containing DMSO-d6 (5% v/v)) The dotted
lines marked in Figure 3(B) indicate the signal shifts of the protons during
change in temperature. Red broken lines in Figure 3(C) indicate the corre-
lations between the protons in GPP (2) and those in the head moiety of
DMPG.
The signals at 7.5 ppm, 8.3 ppm, and 9.1 ppm are assigned
as protons of m-phenyl, o-phenyl and porphyrin -positions, re-
spectively (supporting information). The signal broadening is
wholly due to the existence of LUV macromolecular substance.
At lower temperatures, each signal tends to split into two peaks
(Figure 3B). The splitting occurs around 25–30 C, which is
close to the phase transition temperature of DMPG LUV (vide
infra).22, 23 Consequently, this finding indicates that GPP (2) in-
teracts with DMPG LUV. The signal splitting at low tempera-
tures is caused by the decrease in exchange rate between several
interaction fashions. We also collected the NOESY spectrum of
the mixture of GPP (2) and DMPG LUV at 40 C in order to
address the location of GPP (2) in DMPG LUV (Figure 3C).
Next, we measured UV-vis spectra of GPP in the presence
of amphiphilic systems. The absorbances at ~450 nm or ~685
nm for GPP (2.5 µM) in 10 mM citrate/phosphate buffer (pH
4.2) increased gradually with increasing concentration of SDS
micelles or DMPG LUV (Figure S10). This is indicative of the
interaction between GPP and the interface of those self-assem-
blies. The increase in absorbances shown in Figure S10 is very
similar to the spectral change observed for the pH titration in a
buffer solution (Figure 2A), suggesting GPP in the amphiphilic
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systems undergoes the acid/base equilibrium to generate pM-
Page 6 of 11
1
2
3
4
5
6
7
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GPP form. No significant changes in LUV morphology is con-
firmed by the observation of almost identical distribution in
DMPG LUV diameter (Figure S7). The intensities saturated in
the presence of 8 mM SDS or 1.1 mM DMPG, and the intensity
saturation justifies that all the GPP molecules interacted with
the interface. The red-shift of absorption band from ~446 to 450
nm for pM-GPP form in the self-assembled system suggests GPP
interacted with the self-assembly interface with  lower than the
bulk phase. Therefore, we performed further measurement un-
der these micelle or LUV-interacting intensity saturation condi-
tions to monitor dA-GPP and/or dM-GPP to pM-GPP interconver-
sion for GPP at the self-assembly interfaces.
Figure 4. pH-dependent UV-vis analysis of GPP (2; 2.5 M) at 25 C in 10
mM sodium citrate/sodium phosphate buffer containing DMPG LUV (lipid,
1.1 mM); (A) Spectra measured at pH 4.0, 4.25, 4.50, 4.70, 4.90, 5.05, 5.25,
5.55, 5.90. The change in intensities with decreasing pH are shown in ar-
rows; (B) pM-GPP mole-ratio (XpM-GPP) analyzed from the intensity at ~450
nm (circle, solid lines) and ~685 nm (triangle, broken lines) are plotted
against bulk pH (red). The plots for pure buffer solutions (black) and buffer
containing 31% (w/w) acetone (gray) in Figure 2 are shown for comparison.
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Simultaneous estimation of pH and polarity at the self-as-
sembly interfaces. pH-metric titrations for GPP(2) were per-
formed with a interaction saturated high concentration of
DMPG LUV (lipid, 1.1 mM, Figure 4A) and SDS micelles (8
mM, Figure S11B) at 25 ˚C. As described above, the extinction
coefficient of pM-GPP form does not depends on . The pH-de-
pendent equilibrium pM-GPP mole-ratio (XpM-GPP) at the DMPG
LUV interface was estimated by monitoring absorption inten-
sity at ~450 nm or ~685 nm at the full interaction of GPP with
self-assembled systems according to eq. (2). On the other hand,
for the determination of XpM-GPP value at the SDS micelle inter-
face, only absorbance change at ~685 nm can be used for the
analysis. The higher energy absorption band around 450 nm
tended to gradually blue-shift with decreasing pH from 5.3 to
4.3 and overlapped considerably with that of dA-GPP and/or dM-
GPP (Figures S11B). When using XpM-GPP under different bulk
pH conditions, the interface  was estimated by calculating the
corresponding A419N from the observed intensity at ~420 nm ac-
cording to eq. 3 and 4. The interface  were determined to be
similar at ~61.5 and 60.5 for SDS micelle and DMPG LUV, re-
spectively, at 25 C using eqs. 3 and 4 (Table 1).
The interface pH can be evaluated by monitoring the bulk
pH-dependent pM-GPP mole-ratio between the interface and the
bulk. The increase in the amount of pM-GPP mole-ratios ob-
served in self-assembled systems indicate that the interfaces of
these self-assemblies provide more acidic environments than
the bulk phase does (DMPG LUV: Figure 4; SDS: Figure S11B).
For quantitative measurement of pH at the interface, the pM-
GPP mole-ratio in the absence and presence of interaction satu-
rated high concentration of SDS or DMPG LUV are compared
with that of bulk pH (Figures 4B and S11E). The apparent pH
shift between the interface and the bulk pH () can be estimated
from the difference in pM-GPP ratio between the interface and
the bulk at 25 C, where  should be negative because the inter-
face is more acidic compared to the bulk. Irrespective of a dif-
ferent bulk pH, the  value was estimated to be approximately
–0.54 and –0.59 for DMPG LUV and SDS micelles, respec-
tively (Figures 4 and S11). Because the pH-induced intercon-
version between pM-GPP and dA-GPP and/or dM-GPP is also af-
fected by medium ,  can also be affected by the difference in
 between the interface and the bulk. The polarity contribution
() to  is estimated by the apparent pH shift caused by the dif-
ference in  between the interface and the bulk. The pH devia-
tion (pH) from the bulk to the interface pH (pHinf), and subse-
quently pHinf are obtained from the bulk pH (pHbulk), , and :
Table 1 Temperature- and bulk pH- dependent pH deviation
from the bulk to the interface (pH) and interface dielectric
constant ((i)) for SDS micelle and DMPG LUV
T
Bulk pH
Interface 
pH
(C)
pH =  – 
(5)
(6)
20
25
35
40
20
4.35.3
4.35.3
4.25.2
4.15.1
4.5
63.0  0.2
61.5  0.2
59.4  0.2
57.6  0.2
61.6  0.4
61.5  0.2
0.72  0.03
0.70  0.03
0.69  0.03
0.67  0.03
0.62  0.03
0.62  0.03
pHinf = pHbulk +  – 
SDS
micelle
 is the function of combined interface pH and , whereas  is
related to interface . Conventionally, two separate pH and po-
larity responsive probe molecules are used to evaluate  and ,
respectively.9, 10 However, the change in the locations of these
pH and polarity probes at various interface depths makes the
determination of  erroneous because the interface polarity can
decrease drastically with increasing interface depth from the
water-exposed outer-surface. In contrast, the simultaneous esti-
mation of  and  parameters using a single GPP probe at an
identical interface depth not only enables us to compute the cor-
rect value of  for measuring interface pH, but also provides
simultaneous values of interface pH and .
DMPG
LUV
4.95.3
25
30
35
40
4.5
4.95.3
60.6  0.4
60.3  0.2
0.67  0.03
0.67  0.03
4.5
59.6  0.4
61.9  0.2
0.85  0.03
1.01  0.03
4.95.3
4.5
62.1  0.4
63.0  0.2
1.05  0.03
1.09  0.03
4.95.6
4.5
4.95.6
62.6  0.4
61.4  0.2
1.10  0.03
1.11  0.03
The value of  in acetone/buffer mixed solution was ad-
justed same as the interface  for SDS micelles and DMPG
LUV at 25 C (Table 1), and  was found to be ~0.11 for SDS
micelles and ~0.13 DMPG LUV, respectively, under different
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pH conditions at 25 C (Figure S5 (B, F and G)). With the var-
iation of bulk pH, the estimated pH deviation from the bulk to
1
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3
4
5
6
7
8
the interface (pH) for SDS micelles and DMPG LUV were
found to be similar ~0.70 and ~0.67, respectively, at 25C (Ta-
ble 1, Figures 4B, and S5 (B, F and G)). For anionic self-assem-
blies, the negatively charged headgroup at the interface may in-
volve attractive electrostatic interaction with H+, but it repels
OH. In comparison to the bulk phase, [H+] and [OH] may in-
crease and decrease, respectively, at the interface. However,
[H+] and [OH] remain unchanged in the bulk owing to the
larger volume of the bulk phase than the interface. Therefore,
higher [H+] at the anionic SDS micelle or DMPG LUV interface
than the corresponding bulk phase makes the interface more
acidic compared to the bulk pH.
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Temperature effect on SDS micelle interface pH and polar-
ity. The general temperature effect on interface pH and  were
examined for SDS micelle. Upon increasing the temperature
from 20 C to 40 C, the acid/base pKa at the SDS micelle inter-
face decreased from ~4.8 to 4.6 (Table S2 and Figure S11). At
first, the dependency of interface  on temperature was esti-
mated by analyzing the intensity contribution of dM-GPP from
absorption spectra under various pH conditions according to eq.
1 (for complete deprotonation of GPP, pH > 7.0) or eq. 4 (for a
mixture of protonated and deprotonated GPP, pH < 7.0). Irre-
spective of different bulk pH, the observed intensity (for pH 
7.0) or normalized intensity (A420N for pH < 7.0) at 420 nm in-
creased from ~0.34 to 0.39 with an increase of temperature from
25 C to 40 C (Figure S11), which corresponds to a decrease
of interface  from ~61.5 to 57.6 (Table 1 and Figure S5). Inter-
estingly, the identical decrease of  from 25 C to 40 C was
observed for 31% acetone-containing buffer medium (Table S1,
Figures S4 and S5), suggesting that the typical temperature vs.
 correlations are also maintained at the SDS micelles interface.
In addition, a similar amount of pKa decrease ~0.2 for GPP be-
tween SDS micelle and acetone/buffer medium with  similar
to that of the SDS interface was detected by the increase of tem-
perature from 25 C to 40 C (Tables S1 and S2, Figures S6 and
S11). The results shows that the interface pH for SDS micelle
remains unchanged and the acid/base pKa change for GPP is
caused by the temperature induced variation of interface .
Figure 5. Differential scanning calorimetry (DSC) thermograms of DMPG
LUV (lipids, 1 mM) in 10 mM citrate-phosphate buffer, pH 4.5 (red), pH
4.9 (blue), and pH 5.5 (black).
Temperature- and pH-dependent acid/base equilibrium for
GPP (2.5 µM) were monitored at the DMPG LUV interface to
monitor the effect of phase transition from DMPG gel state to
its liquid-crystalline state on the interface pH and  (Figure
S12). In contrast to SDS micelles, an increase in absorption in-
tensity at ~450 nm and ~685 nm was observed for DMPG LUV
by increasing temperature from 20 C to 40 C (Figure 6 and
S12) under a different, but constant pH. However, major in-
creases in absorption intensity at ~450 nm from ~0.12 to 0.21
and at ~685 nm (from ~0.02 to 0.04) were detected between
25C and 35 C at bulk pH 4.9. No further intensity increase
was observed at above 35 C (Figure 6 and Table 1). Under a
decreased bulk pH condition of 4.5, a major increase in intensity
was noticed at above 30 C up to 40 C (Figure 6B). The in-
crease in Tm for DMPG LUV from 28.0 C to 32.3 C with de-
creasing pH from 4.9 to 4.5 also clearly correlates with the in-
creasing temperature required to obtain the major increase in
pM-GPP mole-ratio from 4.9 to 4.5 (Figure 6). The temperature
induced increase in pM-GPP mole-ratio indicating an increase in
interface acidity can be correlated with the phase transitions
process of DMPG gel state to liquid-crystalline state (Figure 6
and Table 1).
Interface pH/polarity changes during temperature induced
phase transition for DMPG LUV. The gel to liquid-crystalline
phase transitions process of lipids in LUV membrane is not only
affected by temperature, but also dependent on environmental
pH conditions.24 DMPG is known to exhibit weakly energetic
pre-transitions peaks at low temperatures (∼11–15 °C) and
highly cooperative strongly energetic gel to liquid-crystalline
phase transitions (∼23–25 °C).25 The phase transition tempera-
tures (Tm) of DMPG in its LUV were measured by differential
scanning calorimetry (DSC) in 10 mM citrate/phosphate buffer
solution at various pH 4.55.5 (Figure 5). The weak pre-transi-
tion peak at ~16 and 21 °C were observed at pH 5.5 and 5.0,
respectively, although no such peak appeared at pH 4.5. How-
ever, the main phase transition temperature Tm,26 was found to
increase from 28.0 to 32.3 C by decreasing pH from 4.9 to 4.5,
whereas Tm increased to a relatively lower extent from 26.6 to
28.0 C with increasing pH to 5.5.
Figure 6. Temperature dependent UV-vis absorption spectra of GPP (2; 2.5
M) and (inset) corresponding normalized absorption spectrum in 10 mM
sodium citrate/sodium phosphate buffer containing DMPG LUV (1.1 mM)
at (A) pH 4.9 and (B) pH 4.5: black, 20 C; green, 25 C; red, 30 C; blue,
35 C and violet, 40 C. Each spectrum in inset is normalized according to
the similar procedure to obtain normalized intensity (A420N) using eq. (3).
According to our analysis protocol, we first estimated inter-
face  by normalizing each spectrum in Figure 6 and S12. For
example, normalized spectra at pH 4.9 and 4.5 according to eq.
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Page 8 of 11
to 63 was detected for the transition from gel to liquid-crystal-
3 are represented in inset of Figure 6. Interface  for DMPG
LUV at different temperatures and pH values were determined
from the value of normalized intensity (A420N) using eq. 4 (Fig-
ures 6, S12 and Table 1). When DMPG involves a phase transi-
tion process from ~25 C to 35 C at pH 4.9 and ~30 C to 40
C at pH 4.5, the interface  increased from 60.3 to 63.0 and
59.6 to 62.6, respectively (Figures 5, 6 and Table 1). For other
temperatures when DMPG did not involve a phase transition
significantly according to DSC measurements (Figure 5), a de-
crease in interface  with increasing temperature was also ob-
served in similar to SDS micelles (Figure S11, and Table 1 and
S1). Since the acid/base equilibrium for GPP is affected sub-
stantially by environmental  (Figure S5 and Table S1), various
polarity correction factors ( ~0.090.13 depending on different
interface  ~60.363.0) are considered to estimate interface pH
and its deviation from the bulk pH (pH) using eq. 5 and 6. Bulk
pH and temperature-dependent interface pH and pH are listed
in Table 1. A similar increase of pH ~ –0.5 was mostly ob-
served upon increasing temperature from 20 C to 40 C at bulk
pH 4.9 and 4.4, respectively, although the major increase of
pH was observed around the DMPG phase transition temper-
ature (Figures 5 and 6, Table 1). As we observed that pH at typ-
ical self-assembly interface (SDS micelle) does not depend on
temperature, the temperature dependent increase of Stern layer
interface acidity for DMPG LUV should be associated with the
DMPG gel to liquid-crystalline phase transition.
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line phase (inset of Figure 6 and Table 1). Presumably, the lipid
phase transition-induced increase of interface  may be partially
compensated by increasing temperature-dependent decrease in
 (Table 1), resulting in the affection to the small change of in-
terface .
CONCLUSION
A series of UV-vis spectral changes shown by GPP (2) prove
the utility of GPP (2) as a probe for the simultaneous evaluation
of interface pH and polarity for amphiphilic self-assemblies.
Throughout the UV-vis spectral changes during the protona-
tion/deprotonation at inner nitrogen atoms in the porphyrin ring
and aggregation character, GPP (2) is able to reflect the local
pH and polarity in media around GPP (2). Furthermore, the del-
icately balanced structure composed of a porphyrin moiety (hy-
drophobic deprotonated or cationic protonated) and hydrophilic
glucose parts in GPP (2) enable the compound to stay at the
Stern layer of anionic phospholipid self-assemblies. The evalu-
ation of physicochemical properties based on the spectral
changes of GPP (2) in the presence of DMPG LUV is possible
during the phase transition of the lipid self-assembly. The dif-
ference in pKa between the bulk and the lipid membrane surface
is interpretable in terms of structural dynamics of phospholipid
self-assemblies that affects the interface ion penetration abili-
ties. Designed chromophore probes, such as GPP (2), will be
useful to understand chemical events on membrane surface.
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It has been reported that interface pH and  for a charged
amphiphilic self-assembled system are highly controlled by the
packing arrangement of ionic headgroups at the interface Stern
layer.9, 27 A tight headgroup packing arrangement with their low
solvent accessible surface area restricts the electrostatic pene-
tration of H3O+ (for an anionic headgroup) or OHˉ (for a cationic
headgroup) and H2O (for solvation) into the interface stern
layer.9 However, the penetrating ability are gradually improved
with increasing inter-headgroup separation distance (loose
headgroups packing) during gel to liquid-crystalline phase tran-
sition. The packing flexibility may allow the anionic headgroup
to have more access to the bulk water phase for solvation and
penetration of H3O+ into the interface (Scheme 3).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:XXXXXXXXXX.
Synthesis protocol and characterization for GPP(2), several UV-vis
spectra, NOESY spectrum, DLS measurement results and a cryo-
TEM image.
AUTHOR INFORMATION
Corresponding Author
*tmatsuo@ms.naist.jp (T.M.)
*parthaparui@yahoo.com (P.P.P.)
Scheme 3. Schematic representation of lipid headgroup pack-
ing arrangement in DMPG LUV in gel (left) and liquid-crystal-
line (right) phases.
ACKNOWLEDGMENT
T. M. thanks a Grant-in Aid for Scientific Research on Innovative
Areas “Molecular Engine” (JSPS KAKENHI Grant Number
JP19H05395). S. N. also thanks a Grant-in Aid for Scientific Re-
search (C) (JSPS KAKENHI Grant Number JP19K05695). PPP
acknowledges UGC and government of West Bengal for financial
support under RUSA 2.0 scheme (No: 5400-F(Y)). RM and SR
acknowledges UGC for SRF fellowship. We thank Ms. Yoshiko
Nishikawa for mass spectral analyses. We also acknowledge Prof.
Shun Hirota for his kind help in usage of some experimental facili-
ties.
The higher H3O+ penetration at the DMPG LUV interface
makes the interface more acidic by ~0.5 pH unit for liquid-crys-
talline phase compared to the gel phase. Notably, no significant
change of pH at the SDS micelle interface was observed in the
range of 2040 C (Table 1), which surely eliminates any role
of intrinsic temperature induced pH change at LUV interface.
On the other hand, only a small increase of interface  from ~60
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A porphyrin-based probe for simultaneous detection of interface acidity and
polarity during lipid phase transition of vesicle
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Graphical Abstract
Estimation of pH and polarity changes at an identical interface depth of membrane during gel to
liquid-crystalline phase transition of lipid using a glucose-pendant porphyrin molecule.
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