Optimisation of two-photon induced cleavage of molecular linker systems for drug delivery

Article abstract of DOI:10.1016/j.jphotochem.2009.12.016

Photoreactive linker systems for drug delivery in modern ophthalmology are crucial for the efficiency of such applications. We compare 5 different linker molecule candidates (truxillic acid and 4 dimers of coumarin derivates) in solution regarding their chemical stability and two-photon induced cleavage efficiency, and shed light into the role of molecular structure in the cleavage reaction. Dimers of the coumarin family showed much higher two-photon induced cleavage efficiency, achieving the highest reaction cross-section when tert-Butyldimethylsilyl (TBS) was used as a substituent, being almost 5-times higher than the unsubstituted coumarin dimer. Coumarin-based linker systems react promptly with nucleophilic solvents, in particular with short chain alcohols. The polarity of such solvents which do not cause a lactone ring opening seems not to influence the cleavage of the cyclobutane ring in coumarin dimers.

Full text of DOI:10.1016/j.jphotochem.2009.12.016

Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 188–192
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Optimisation of two-photon induced cleavage of molecular linker systems for
drug delivery
T. Buckupa, A. Southana, H.-C. Kima, N. Hamppa, M. Motzkusb,∗
a Physikalische Chemie, Philipps-Universität Marburg, D-35043 Marburg, Germany
b Physikalisch-Chemisches Institut, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 229, D-69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Photoreactive linker systems for drug delivery in modern ophthalmology are crucial for the efficiency
of such applications. We compare 5 different linker molecule candidates (truxillic acid and 4 dimers
of coumarin derivates) in solution regarding their chemical stability and two-photon induced cleavage
efficiency, and shed light into the role of molecular structure in the cleavage reaction. Dimers of the
coumarin family showed much higher two-photon induced cleavage efficiency, achieving the highest
reaction cross-section when tert-Butyldimethylsilyl (TBS) was used as a substituent, being almost 5-
times higher than the unsubstituted coumarin dimer. Coumarin-based linker systems react promptly
with nucleophilic solvents, in particular with short chain alcohols. The polarity of such solvents which do
not cause a lactone ring opening seems not to influence the cleavage of the cyclobutane ring in coumarin
dimers.
Received 22 September 2009
Received in revised form 8 December 2009
Accepted 15 December 2009
Available online 23 December 2009
PACS:
33.00.00
82.50.−m
Keywords:
Photo-induced cleavage
Reaction quantum yield
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
lasers with emission in the visible can be easily used as two-photon
excitation sources [6].
New drug delivery methods are a crucial aspect in several fields
of modern medicine because they promise precise targeting of
patients’ diseases and optimised administration. Novel technolo-
gies based on gene therapy, cyclodextrins and lipid vesicles, just to
mention a few, enabled new ways of overcoming drug absorption
barriers in the skin and the mucosa [1]. Particularly in ophthalmol-
ogy, a new photochemical drug delivery system has been recently
suggested which is rapidly developing to become a valuable non-
invasive therapeutic tool in the treatment of secondary cataract
[2–4]. In this new device, the drug delivery is based on the photo-
induced cleavage of a linker system which immobilizes the drug to
an artificial drug-loaded polymeric intraocular lens (IOL).
An important aspect of this concept is the excitation mechanism
of the linker system. In order to reach precise temporal targeting
at the beginning of secondary cataract, the linker system must not
be cleaved upon exposition to sunlight, which cannot be avoided in
the eye. Two-photon excitation can be used with unchallengeable
advantages: Multiphoton processes require high peak-intensities,
which makes the drug release specific not just in time but also gives
a high spatial resolution to the method [5]. When combined with
linker systems that absorb in the UV spectral region, commercial
In order to achieve an effective treatment, i.e. a sufficient quan-
tity of the drug released within a short irradiation time, an efficient
linker system must be found. In previous works, we have explored
coumarin derivatives as linker systems [2,7]. Coumarin dimers in
solution show a two-photon absorption cross-section of around
1 GM and a reaction quantum yield of about 20–23%. In this con-
tribution we investigate and compare systematically the cleavage
efficiency of 5 linker systems (Fig. 1). Firstly, we compare two
linkers based on different molecular families, truxillic acid (I) and
unsubstituted coumarin dimer (II). Secondly, we investigate the
effect of different substituents in 7-position of coumarin dimers on
the total reaction cross-section. For this purpose we compare com-
pounds III and V to the unsubstituted compound II. Thirdly, the
effect of methylation of the cyclobutane ring in 4-position on the
total reaction cross-section is investigated by comparing III and IV.
2. Experimental
2.1. Sample preparation
All dimer molecules were synthesized as described in Ref.
[2,8,9] and used after HPLC purification. Acetonitrile (Fluka, for
UV-spectroscopy), ethanol (Sigma Aldrich, absolute), methanol
(Fisher Scientific, HPLC grade) and isopropanol (Acros Organics,
HPLC grade) were used without further purification. Acetonitrile
∗
Corresponding author. Tel.: +49 6221 54 5042; fax: +49 6221 54 5050.
E-mail address: Marcus.Motzkus@pci.uni-heidelberg.de (M. Motzkus).
1010-6030/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2009.12.016

T. Buckup et al. / Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 188–192
189
Fig. 1. Structures of compounds I–V and respective monomers. (I) Truxillic acid,
(II) coumarin dimer, (III) tert-Butyldimethylsilyl (TBS) – coumarin dimer, (IV) tert-
Butyldimethylsilyl (TBS) –4-methyl-coumarin dimer, (V) acetoxycoumarin dimer.
was selected as solvent and used in the irradiation studies for all
linker systems (Section 2.3). Approx. 10 mg of each compound was
dissolved in 10 mL acetonitrile yielding concentrations of about
2–3 mol/L. In order to calculate the concentration changes and the
reaction cross-sections, the molar absorption coefficient was deter-
mined for all 5 compounds and is summarized in Table 1.
2.2. Irradiation setup
The irradiation study was performed using an Nd-YAG laser
(Coherent, Infinity) with emission at 532 nm. Typical intensities
between 3.6 and 6.5 W (at 100 Hz repetition frequency) with pulse
durations of 3 ns were used for all linker systems. The pulse form
was carefully checked for possible artefacts with a fast photodiode.
The irradiation volume is defined by laser beam area and cell thick-
ness. In our case it was typically 40 ␮L, a factor 63 smaller than the
typical sample volume. In order to maintain a homogenous concen-
tration while the reaction proceeds, a magnetic stirrer was used
Fig. 2. Linear absorption spectra of compounds I–V (continuous line) and respective
monomers (traced line) measured in acetonitrile.
Table 1
during the measurements. For the coumarin dimers (compounds
II–V), the linear absorption change at the absorption maximum
around 320 nm was used to follow the photo-induced cleavage,
because dimer and monomer absorption are well separated and
do not interfere with each other at this wavelength (Fig. 2). In case
of compound I, the dimer and monomer absorption overlap one
another. However, the absorption coefficient of the dimer is about
a factor 1000-times smaller than that of the monomer, justifying
the use of the absorption change at 272 nm in this case.
In order to improve the absorption measurement precision, the
duration of irradiation was chosen so that the absorption change at
the observed wavelength sums up to 0.20–0.30 OD. For example,
for compound I the irradiation time was 4 h, while for compound
V it was less than 1 h.
Molar absorption coefficients for compounds I–V in acetonitrile.
Compound
ꢀ (nm)
ε (L/mol cm)
Monomer
I
II
III
IV
V
272
310
317
314
310
19,965
5,170
13,845
14,664
8,309
Dimer
I
II
III
IV
V
259
272
276
276
260
511
3,686
4,067
4,456
3,557

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T. Buckup et al. / Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 188–192
2.3. Chemical stability
The determination of the two-photon cleavage reaction effi-
ciency requires accurate knowledge of laser parameters and sample
concentrations before and after the photochemical reaction. In
this context, an instable photoproduct or reactant may interfere
with the calculation of reaction and two-photon cross-sections.
Coumarin dimers tend to hydrolyze the lactone ring by nucleophilic
attack as found previously for other derivates [10]. For this reason
methanol and other nucleophilic solvents must not be used in such
measurements. Acetonitrile is a solvent for all investigated com-
pounds and also no lactone ring cleavage has been observed. For
these reasons acetonitrile was used throughout all the experiments
as a solvent.
3. Results and discussion
The two-photon induced cleavage was followed by recording
the absorption difference before and after irradiation. The quantifi-
cation of the reaction and two-photon absorption cross-section was
performed as in [11,12]. The reaction cross-section ˝2PA is given by
the product of the two-photon absorption cross-section ꢁ2PA and
the reaction quantum yield ꢂ2PA
:
Fig. 3. Absorption change for compounds (a) I and (c) II. The evolution of the absorp-
tion change with the irradiation time is shown in (b) for compound I and in (d) for
compound II.
˝
2PA
= ꢁ2PA × ꢂ2PA
.
(1)
The quantum yield was obtained from one photon irradiation
for compound III and, due to almost constant values for previously
investigated coumarin dimers, was also assumed to be constant
here. This assumption is based on the experimental observation
that the linear absorption spectrum scales perfectly with the wave-
length dependence of the two-photon induced reaction [7]. Similar
assumption was made for the quantum yield of truxillic acid [13].
The results are summarized in Table 2.
For compounds I and II, the absorption change upon irradiation
is exemplarily shown in Fig. 3. The two-photon cleavage efficiency
of the coumarin dimer exceeds that of truxillic acid (I) by a factor
30. With similar experimental irradiation parameters, compound
I requires almost 5 h of irradiation to achieve just one third of the
turnover achieved with compound II in 30 min of irradiation. This
strong difference must be caused by the two-photon absorption
cross-section, since the quantum efficiency for the coumarin dimer
photocleavage is a factor 2 better than for the truxillic acid [13].
This means that the ꢁTPA for truxillic acid is still a factor 11 worse
than for compound II.
Compounds III and IV share the same substituent in 7-position
but differ by the presence of two methyl groups on the cyclobutane
ring. In the past we speculated that due to deformation in the ring
caused by the methyl substituents, compounds with this feature
should be cleaved easily and thus allow a more efficient overall
photo-induced reaction. They did not fulfil this expectation, but
both compounds showed within our precision limits similar reac-
tion cross-sections. The most important molecular feature for the
reaction efficiency is the substituent in 7-position. All substituted
coumarin compounds (III–V) show a higher reaction cross-section
˝
than the unsubstituted coumarin compound II. In particu-
2PA
lar compounds III-IV, which share the same substituent, exhibit a
reaction cross-section about 5-times higher than compound II.
For non-centrosymmetric molecules in the two-states approx-
imation, the TPA depends on the strength of transition dipole
moment and the dipole moment changes between the excited and
ground state [14,15]. Coumarins are molecules with a first excited
state S1 showing a strong intramolecular charge transfer (ICT) char-
acter, which implies a much larger dipole moment than in the
S0 [16]. The symmetric arrangement of the carboxyl and phenyl
groups in truxillic acid suggests a higher quadrupole than a dipole
character. The transition dipole moment of this dimer is so small
that single-photon absorption is negligible compared to cinnamic
acid (monomer of compound I) (see Fig. 1). Although it is known
for ␲-conjugated systems that chain length extension results in an
increase of the transition dipole moment, the effect of dimerization
on the ICT character for the S1-state of the coumarin monomer is
unknown at present. Despite of that, the ICT property of the S1-
state of coumarins together with the sensible strong quadrupolar
moment of truxillic acid may be an explanation for the much bigger
TPA cross-section of coumarin dimers compared to truxillic acid.
In ␲-conjugated systems, the TPA cross-section can usually
be optimised using electron donor and acceptor groups. TPA of
coumarin dimers can be enhanced in a similar way. For exam-
ple, the carbonyl groups in compounds II–V act as electron donor
while substituents with different electron donor/acceptor qualities
can be added. The variation of the reaction cross-section observed
Table 2
The pulse energy Epulse, dimer initial concentration D0, monomer concentration variation (ꢃCmono) with the respective irradiation time, cleavage cross-section (˝2PA),
photo-induced cleavage yield (ꢂ2PA) and absorption cross-section (ꢁ2PA) for two-photon induced reaction [1 GM (Göppert Meyer) = 10−50 cm4 s photon−1].
Compound
Epulse (mJ)
D0 (␮M)
ꢃCmono (␮M)
˝
(GM)
ꢂ2PA (%)
ꢁ2PA (GM)
2PA
I
II
65
66
36
37
35
36
2.0
2.5
1.6
1.6
1.7
3.7
8 [4.5 h]
2.69 × 10−3
0.05 0.1
0.23 0.03
0.24 0.01
0.22 0.03
0.09 0.01
12.3
23.1
23.1
23.1
23.1
23.1
0.02 0.01
0.22 0.02
1.01 0.02
1.02 0.03
0.97 0.11
0.37 0.01
25 [30 min]
16 [30 min]
18 [30 min]
15 [30 min]
24 [45 min]
III (in MeCN)
III (in iPrOH)
IV
V

T. Buckup et al. / Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 188–192
191
This result is an additional information about the reaction mech-
anism and the chemical stability of the coumarin dimer in polar
environments. Hasegawa and co-workers investigated the pho-
tochemical behaviour of several dimers of coumarin derivates,
focusing on the cleavage reaction pathway after single photon
excitation [10]. Depending on the coumarin derivate, nucleophilic
solvents like alcohols lead to asymmetric fission of the cyclobutane
ring, giving two different species (not the monomer). Thus, since in
our case the reaction cross-section for both solvents was the same,
we can safely affirm that cleavage of compound III, the compound
with the most effective reaction, evolves over a symmetric reaction
pathway in these two solvents as confirmed by HPLC analysis. This
is of importance in technical applications if the reverse reaction,
i.e. dimerization, is to be exploited as, for example, in optical lenses
with tuneable refractive properties based on these systems [20].
4. Conclusions
The investigation of photo reactive linker systems for drug deliv-
ery in ophthalmology is a crucial step in the optimisation of this
new technology. Linker systems must be chemically stable and
present a high reaction cross-section for efficient implementation.
In this work we have investigated several linker systems for poten-
tial use in a drug delivery method dedicated to ophthalmology.
Using a two-photon induced process, we systematically character-
ized the two-photon reaction cross-section of 5 different systems.
Dimers from the coumarin family showed the highest photo-
induced cleavage efficiencies. Furthermore, we discovered that
substituents modify strongly the reaction cross-section, improving
the photoreaction almost by a factor 5. Complementing previous
results for the dimerization of coumarin molecules in solution, the
dimer cleavage seems to evolve independent of the environment
when non-nucleophilic solvents are used in order to assure chem-
ical stability.
Fig. 4. Investigation of the cleavage reaction efficiency of compound III for two
solvents (a) acetonitrile (open squares with continuous line) and isopropanol (filled
circles with traced line).
for compounds II–V is compatible to complementary experimental
observations [17] and theoretical calculations [16,18] for coumarin
monomers. In Ref. [16], the transition dipole moment was shown
to scale with the degree of alkylation of aminocoumarin derivates.
The TPA cross-section increased upon increasing the number of
alkyl groups in Coumarin 120, Coumarin 1 and Coumarin 480. Even
for trifluorinated coumarins, the calculated TPA follows the same
alkylation trends found for the aminocoumarins. In case of the
compounds investigated in this work, compound I does not have
any alkyl substituents and therefore showed the smallest reaction
efficiency. Compounds III and IV, which share very similar struc-
tures and a higher degree of alkylation than the other compounds,
showed the highest reaction efficiency. Compounds III and IV have
three times more alkyl groups than compound V but the increase
of the TPA cross-section was smaller (about a factor 2–3). From this
trend, it seems that the TPA cross-section scales linearly with the
number of alkyl groups, suggesting that alkylation is also an impor-
tant control knob in the two-photon induced cleavage reaction
even if the alkylation originates from a tert-Butyldimethylsilyl (TBS)
group. It is important to note that two-photon absorption cross-
sectionsmay be slightlymodifiedwhenmeasured with nanosecond
pulses [19]. Such pulses can re-pump excited state populations and
thereby achieve higher reaction turnovers, as we observed in a
previous work [7].
Acknowledgement
Financial support of this project (FKZ 13N8978) was provided
by the German Ministry of Education and Research (BMBF).
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