Temperature responsive phosphorescent small unilamellar vesicles

Article abstract of DOI:10.1039/c2cc33279e

Self-assembled lipid vesicles with embedded amphiphilic terbium(iii) complexes show a strong temperature dependence of their phosphorescence intensity and lifetime in the physiological range.

Full text of DOI:10.1039/c2cc33279e

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Temperature responsive phosphorescent small unilamellar vesiclesw
Mouchumi Bhuyan and Burkhard Koenig*
Received 7th May 2012, Accepted 7th June 2012
DOI: 10.1039/c2cc33279e
Self-assembled lipid vesicles with embedded amphiphilic terbium(III)
complexes show a strong temperature dependence of their phosphor-
escence intensity and lifetime in the physiological range.
Temperature is a fundamental physical property of matter
important in everyday life, in scientific and industrial applications.
The methods to measure temperature can be divided into
two main techniques: contact thermometry and non-contact
thermometry. Contact methods use thermocouples, thermistors,
and resistance temperature detectors (RTDs), whereas non-
contact methods often use the spectral emittance of a material
for readout.1
Non-contact optical techniques have various advantages in
terms of sensitivity and real-time monitoring over a wide
range, from femtoseconds to seconds.2 Among the available
optical methods, infrared thermometry that uses the principle
of blackbody radiation is flexible and easy to use, but can only
measure the temperature of surfaces, thus limiting its applications.3
Therefore luminescence based optical sensors have attracted
much attention, because of their fast response, high spatial
Fig. 1 Schematic representation of LNT.
resolution, accurate usability even in strong electromagnetic
fields4 and safety of remote handling.5 Such sensing probes
while Tb3+ complexes are less explored. One reason is their
use appropriate luminophores ranging from polycyclic
smaller luminescence dependence on temperature.9 However,
the longer luminescence lifetime of the Tb3+ ion compared to
Eu3+ makes Tb3+ complexes the favoured systems for the
lifetime based optical sensing of temperature.10 Having this in
mind we used the diethylenetriamine pentaacetic acid (DTPA)
based amphiphilic Tb3+ complex (Tb-1) as a probe for the
optical sensing of temperature. By embedding the complex
[for detailed experimental procedures and analytical data of
the prepared compounds, see ESIw] in different phospholipids
we obtain reversible thermosensitive luminescent vesicles
(Fig. 1) at an adjustable temperature range. The luminescent
nanosized vesicles (LNT) with embedded Tb3+ complexes
were prepared by the previously reported film-hydration
method.11 The multilamellar vesicles were extruded through
a membrane yielding unilamellar vesicles of 90–100(ꢀ5) nm in
size (see Fig. S11 and S12 in ESIw). We have used different
commercially available phospholipids for the preparation of
LNT 1–6 (Table 1).
aromatic hydrocarbons to metal complexes.2 Temperature
sensitive probes were incorporated into nanogels for intra-
cellular thermometry,6 and in sensor films or thermo-sensitive
polymers to allow spatially resolved temperature imaging.7
Recently tripositive lanthanide complexes have been used
for optical sensing of temperature.4,7,8 The use of such lanthanide
complexes has several advantages over organic luminophores:
(a) they have long emission wavelengths, (b) lanthanide emis-
sions are Laporte forbidden and are therefore characterized by
very long luminescence lifetimes, which is useful for applica-
tions in biological media by excluding background emission
using time delayed detection, (c) 4f electrons of the lanthanides
are highly shielded from the chemical environment by the 5s
and 5p orbitals thus making them insensitive to the environ-
ment. Among the tripositive lanthanides, typically Eu3+ based
complexes have been used as optical temperature sensors,
Institut fur Organische Chemie, Universitat Regensburg, Regensburg,
¨
¨
The emission intensities of lanthanide complexes depend on
their concentration.13 Therefore several different concentra-
tions of Tb-1 were used in the lipid membranes and in terms of
emission intensity, 5 mol% of Tb-1 with respect to the used
lipids is the most suitable condition for these systems.
93040, Germany. E-mail: burkhard.koenig@chemie.uni-regensburg.de;
Fax: +49-941-943-1717; Tel: +49-941-943-4575
w Electronic supplementary information (ESI) available: Experimental
details, characterisation of synthesized complexes and vesicles,
temperature dependent lifetime and emission intensity data of the
vesicles. See DOI: 10.1039/c2cc33279e
c
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Chem. Commun., 2012, 48, 7489–7491 7489

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Table 1 Composition response range of LNT 1–6
Most
Effective sensitive
range (1C) rangeb (1C) (% 1Cꢁ1
Phospholipids
Vesicles used [Tm (1C)]
Sensitivityb
a
)
LNT1
LNT2
LNT3
LNT4
LNT5
18 : 0 PC (DSPC) [55] 0–60
25–55
20–40
10–23
25–55
n.a.
ꢁ3.5
ꢁ3.0
ꢁ4.2
ꢁ1.8
n.a.
16 : 0 PC (DPPC) [41] 0–40
14 : 0 PC (DMPC) [23] 0–25
DSPE-PEG350 [B60] 0–60
18 : 1c9 PC (DOPC) n.a.
[ꢁ20]
LNT6
PC(18 : 0/14 : 0)
(SMPC) [30]
0–30
15–30
25–55
ꢁ2.8
ꢁ1.4
LNT4c + DSPE-PEG350 [B60] 0–60
BSA
a
b
Tm = phase transition temperature of lipid. The most sensitive
temperature range and sensitivity per 1C are based on the temperature
dependent lifetime measurements carried out in HEPES buffer at pH 7.4.
Fig. 3 Reversibility of luminescence intensity of LNT3 in the measured
temperature range (changes at 545 nm emission wavelength are shown).
c
Non-specific interactions between the constituent lipids and BSA12 are
an increase in temperature and each system shows more than
85% decrease in luminescence intensity around the phase
transition temperature of the constituent lipid used (Fig. 2).
The emission changes for the vesicular systems, LNT 1–6
(except LNT5), are reversible within the measured temperature
range, as exemplarily shown in Fig. 3 for LNT3.
minimised by shielding the vesicular surface using DSPC-PEG350 having
oligoethylene glycol residues attached to its polar head groups.
All vesicles (LNT) containing the Tb3+ complex exhibit a
phosphorescence emission intensity at 488, 545, 584, 620 nm,
corresponding to the electronic transition from the 5D4 state to
However the emission intensity based measurements often
suffer from changing sensor concentrations or drifts of the
optoelectronic systems (lamps and detectors),8c whereas decay
lifetime based data are free from these drawbacks and are
more reliable.8c The synthesised luminescent vesicular systems,
LNTs, also show reversible changes in the decay lifetimes with
varying temperature. The luminescence decay vs. time curve
follows a first order exponential decay at every measured
temperature (see Fig. S4 in ESIw) and the average lifetime
(t) is determined by eqn (1),
7
7
7
7F3, F4, F5, F6 states of the Tb3+ ions. However, at 25 1C,
the emission intensity is significantly different for LNT1 (DSPC
lipids, Tm = 55 1C) and LNT5 (DOPC lipids, Tm = ꢁ20 1C)
(see Fig. S1 in ESIw) which indicates that the phase transition
temperature of the lipids affects the emission intensity.
The temperature dependent emission intensity and lifetime
measurements of the vesicular systems LNT were carried out
mainly in HEPES buffer at pH 7.4. There is no significant
effect on the emission temperature response by pH changes in
the range from pH 6 to 8, by changing the buffer substance or
varying the salt concentration (for details see ESIw, Fig. S7
and S8). All vesicles show a decrease in emission intensity with
y = A1*exp(ꢁx/t) + y0 (1)
where y = lifetime at time x; y0 = initial lifetime; x = time;
t = 1/(decay constant); A1 = pre-exponential factor.
The lifetime vs. temperature plot shows that for every LNT,
average lifetime (t) decreases with an increase in temperature
and the system shows high sensitivity around the phase
transition temperature (Tm) of the constituent lipid (Fig. 4).
Thus the most sensitive temperature range for these nanosystems
can be tuned by changing the constituent lipids (Table 1).
The luminescence intensity and lifetime of lanthanides can
be affected by many factors. The overall decay rate constant of
the luminescence level can be expressed by the following
equation,14 where kr is the radiative rate constant and knr
and knr(T) are the non-radiative temperature independent and
temperature dependent decay rate constants.14
1
k ¼ ¼ kr þ knr þ knrðTÞ
ð2Þ
t
The non-radiative transition consists of the vibrational excita-
tion, back energy transfer (BET) to the triplet excited state of
the attached sensitizer from the 5D4 state of Tb(III) and the
concentration quenching. The temperature-dependence of the
decay rate constant is expected for the vibrational excitation
5
and BET in the non-radiative transition from the D4 level of
the terbium(III) ion.9a A decrease in luminescence intensity can
Fig. 2 Temperature dependence of emission intensity measurements
for LNT3 [top] and LNT4 [bottom] [lex = 285 nm; conc. of Tb(III) =
[5 ꢂ 10ꢁ6 M].
therefore be related to both of these factors, while the decrease
c
7490 Chem. Commun., 2012, 48, 7489–7491
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complexes. These aggregates find applications in sensing and
imaging of physiological temperatures by observing changes
of their phosphorescence intensity and lifetime. The most
sensitive temperature and sensitivity per 1C can be tuned by
merely varying the phase transition temperature of the
constituent lipids as demonstrated by several examples. The
membranes can be processed, e.g. by simple spreading on a
glass surface without losing their sensing ability. Since Tb(III)
has a very long luminescence lifetime, possible interference of
background emission arising from proteins or cells can be
avoided by delayed readout.
MB thanks the Bavarian Research foundation for graduate
fellowship. We thank the University of Regensburg and the
Deutsche Forschungsgemeinschaft for support of our research.
Fig. 4 Average lifetime (ꢀs.d) (ms) vs. temperature (1C) plot for LNT3.
Notes and references
in luminescence lifetime depends only on the energy dissipa-
5
tion from the D4 state of Tb(III). These facts give a rationale
1 (a) R. E. Bentley, Temperature and Humidity Measurement, Springer,
Singapore, 1998; (b) http://www.temperatures.com.
2 M. Mitsuishi, S. Kikuchi, T. Miyashita and Y. Amao, J. Mater.
Chem., 2003, 13, 2875.
3 E. F. J. Ring, Infrared Phys. Technol., 2007, 49, 297.
4 S. M. Borisov and O. S. Wolfbeis, Anal. Chem., 2006, 78, 5094.
5 R. Schorer, E. Friess, K. Eberl and G. Abstreiter, Phys. Rev. B,
1991, 44, 1772.
6 S. Uchiyama, A. Prasanna de Silva and K. Iwai, J. Chem. Educ.,
2006, 83, 720.
7 G. E. Khalil, K. Lau, G. D. Phelan, B. Carlson, M. Gouterman,
J. B. Callis and L. R. Dalton, Rev. Sci. Instrum., 2004, 75, 192.
8 (a) H. Lam, G. Rao, J. Loureiro and L. Tolosa, Talanta, 2011,
84, 65; (b) Y. Ohishi and S. Takahashi, Appl. Opt., 1986, 25, 720;
(c) H. Peng, M. I. J. Stich, J. Yu, L. Sun, L. H. Fischer and
O. S. Wolfbeis, Adv. Mater., 2010, 22, 716; (d) Y. Dwivedi and
S. B. Rai, Sens. Actuators, A, 2010, 163, 37; (e) H. Sakaue,
C.-Y. Huang and J. P. Sullivan, Sens. Actuators, B, 2011, 155, 372.
9 (a) S. Katagiri, Y. Tsukahara, Y. Hasegawa and Y. Wada, Bull.
Chem. Soc. Jpn., 2007, 80, 1492; (b) L.-N. Sun, J. Yu, H. Peng,
J. Z. Zhang, L.-Y. Shi and O. S. Wolfbeis, J. Phys. Chem. C, 2010,
114, 12642.
10 (a) M. D. Chambers and D. R. Clarke, Surf. Coat. Technol., 2007,
202, 688; (b) D. R. Clarke and M. M. Gentleman, Surf. Coat.
Technol., 2007, 202, 681.
11 J. Lasch, V. Weissig and M. Brandl, in Liposomes, ed. V. Torchilin
and V. Weissig, Oxford University Press, 2nd edn, 2003.
12 O. D. Velev, Adv. Biophys., 1997, 34, 139.
for the sensitivity of LNT near the transition temperature of
the lipid. The constituent phospholipids are in a tightly packed
gel phase below the transition temperature (Tm)15 leading to
deactivation of the vibrational excitation and hence a longer
lifetime of the embedded Tb(III) ion. Near or above the phase
transition temperature, the lipid bilayers are more or less in the
liquid crystalline phase15c with higher chain mobility of the
constituent lipids resulting in a drastic decrease in the lumi-
nescence lifetime by vibrational dissipation of energy from the
5D4 state of the Tb(III) ion. Above Tm of the corresponding
lipids, the nanosystems show negligible sensitivity towards an
increase in temperature. The phase transition temperature is
not affected by pH changes in the range from pH 6 to 8
allowing the application of the system in this range.
The fully PEGylated vesicle, LNT4, shows reversible change
in emission intensity with temperature. However, for a given
wavelength, the intensity vs. temperature plot is convex for the
non-PEGylated vesicles, while it is concave for the completely
PEGylated vesicular system, LNT4 (Fig. 2 (bottom). The
difference in the nature of the plot can be attributed to
increased chain mobility of the PEGylated lipids as shown
by Belsito et al. using spin-label ESR measurements.16 The
temperature dependent luminescence changes of LNT4 are not
affected by the presence of bovine serum albumin (BSA, Table 1).
The Tb-functionalized vesicles are effective in regular cell
culture medium (DMEM, 10%FCS), however, the presence
of phenol red in standard solutions interferes with the absorption
of the dyes and thereby lowers the emission intensity.
13 (a) D. de Graaf, S. J. Stelwagen, H. T. Hintzen and G. de With,
J. Non-Cryst. Solids, 2003, 325, 29; (b) J. Holsa, M. Leskela and
¨
L. Niinisto, Mater. Res. Bull., 1979, 14, 1403.
¨
¨
¨
14 N. Sabbatini, M. Guardigli and J.-M. Lehn, Coord. Chem. Rev.,
1993, 123, 201.
15 (a) J. H. Crowe, A. E. Oliver, F. A. Hoekstra and L. M. Crowe,
Cryobiology, 1997, 35, 20; (b) W. F. Wolkers, H. Oldenhof and
B. Glasmacher, Cryobiology, 2010, 61, 108; (c) M. Caffrey and
J. Hogan, Chem. Phys. Lipids, 1992, 61, 1.
In summary, we have developed a nanosized luminescent
thermometer by self-assembly of lipids and amphiphilic Tb(III)
16 S. Belsito, R. Bartucci, G. Montesano, D. Marsh and L. Sportelli,
Biophys. J., 2000, 78, 1420.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 7489–7491 7491