Two-photon absorption triggered drug delivery from a polymer for intraocular lenses in presence of an UV-absorber

Article abstract of DOI:10.1016/j.jphotochem.2012.08.012

Two-photon-absorption (TPA) triggered photochemistry is a versatile tool to photochemically release compounds in an uncaging reaction where the desired reaction should occur behind an UV absorbing barrier, e.g. as in intraocular lenses (IOLs). Nonlinear effects of the UV-absorbing barrier, in this case the cornea, are negligible as long as the laser light passes through it at an intensity low enough that nonlinear effects do not occur. Only in the focus of the beam, i.e. inside the IOL, the required intensities are reached and TPA triggered photo cleavage occurs. The situation becomes complicated as soon as UV-absorbers are admixed to the polymer material. We show that typical concentrations of an UV-absorber only slightly affect the TPA-triggered uncaging reaction rate. As a testbed we used a newly synthetized acrylic polymer derivatized with an o-nitrobenzyl linker group carrying 5-fluorouracil as the model drug to be released. No photochemical decomposition of the UV absorber was observed. The release rate of 5-fluorouracil in presence of the UV absorber was reduced by about 6% only. Further the polymer presented here, is the first to release unmodified 5-fluorouracil without any auxiliary groups attached. This is important for potential applications in humans.

Full text of DOI:10.1016/j.jphotochem.2012.08.012

Journal of Photochemistry and Photobiology A: Chemistry 248 (2012) 8–14
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Two-photon absorption triggered drug delivery from a polymer
for intraocular lenses in presence of an UV-absorber
Daniel Kehrloessera, Philipp J. Behrendta, Norbert Hamppa,b,∗
a Faculty of Chemistry, University of Marburg, Hans-Meerwein-Str. Bldg. H, D-35032 Marburg, Germany
b Materials Sciences Center, University of Marburg, Hans-Meerwein-Str. Bldg. H, D-35032 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2011
Received in revised form 15 August 2012
Accepted 23 August 2012
Available online 31 August 2012
Two-photon-absorption (TPA) triggered photochemistry is a versatile tool to photochemically release
compounds in an uncaging reaction where the desired reaction should occur behind an UV absorbing
barrier, e.g. as in intraocular lenses (IOLs). Nonlinear effects of the UV-absorbing barrier, in this case the
cornea, are negligible as long as the laser light passes through it at an intensity low enough that nonlinear
effects do not occur. Only in the focus of the beam, i.e. inside the IOL, the required intensities are reached
and TPA triggered photo cleavage occurs. The situation becomes complicated as soon as UV-absorbers are
admixed to the polymer material. We show that typical concentrations of an UV-absorber only slightly
affect the TPA-triggered uncaging reaction rate. As a testbed we used a newly synthetized acrylic polymer
derivatized with an o-nitrobenzyl linker group carrying 5-fluorouracil as the model drug to be released.
No photochemical decomposition of the UV absorber was observed. The release rate of 5-fluorouracil in
presence of the UV absorber was reduced by about 6% only. Further the polymer presented here, is the
first to release unmodified 5-fluorouracil without any auxiliary groups attached. This is important for
potential applications in humans.
Keywords:
ortho-Nitrobenzyl
Single-photon-absorption
Two-photon-absorption
5-Fluorouracil
Drug delivery
UV-absorber
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
800-thousand times per year in Germany alone. Most often the
cause is cataract, which according to the WHO is responsible of 33%
The photoactivity of ortho-nitrobenzyl compounds was
described first by Silber and Ciamician [1]. In the 1960s they
were introduced as photolabile protective groups into organic
synthesis [2–4]. The concept of caged compounds, meaning the
deactivation of biological compounds by derivatization with this
photolabile protective group, was introduced in the late 1970s
[5,6]. Today ortho-nitrobenzyl based caged compounds are used
mainly for biophysical and biochemical investigations [7–10].
The mechanism of the single photon absorption (SPA) induced
photo release process (Fig. 1) is quite well understood, as is the
influence of additional substituents [11–15]. The ability to release
a therapeutic payload from a carrier system is the major goal of
drug delivery.
of visual impairment worldwide [16]. Triggered drug release from
implanted IOLs is a novel approach to treat secondary cataract, a
common post-operative complication occurring in 10–50% of the
cases within 3–5 years [17,18]. Secondary cataract is an opacifi-
cation caused by the proliferation and the migration of retained
lens epithelial cells into the visual axis. Several photoactive mate-
rials have been developed to provide photochemical drug delivery
from IOLs, matching the required material properties like trans-
parency, two-photon activity and optimized drug diffusion. In all
these systems drug attachment to the polymer moiety is achieved
through [2 + 2] cycloaddition. This photochemical step in the syn-
thesis of the materials causes low yields or extensive purification
steps [19–26].
Our interest in photochemical drug delivery is focused on
intraocular lenses (IOLs) which are implanted into the eye about
In technical applications of acrylic polymers UV absorbers are
integral compounds of the polymers on the market. The lightfast-
ness of acrylic polymers is dramatically decreased without such
additives and tanning is observed even after exposure to small UV
light doses. For intraocular lenses the UV absorber requirement
is regulated in order to protect the posterior of the eye from UV
exposure.
As both UV absorber and photo cleavable drug-linker are uni-
formly distributed within the bulk polymer there is a potential
conflict between the desired therapeutic photo cleavage of the
∗
Corresponding author at: Materials Sciences Center, University of Marburg,
Hans-Meerwein-Str. Bldg. H, D-35032 Marburg, Germany. Tel.: +49 6421 282 5775;
fax: +49 6421 282 5798.
E-mail addresses: daniel.kehrloesser@basf.com (D. Kehrloesser),
behrendp@staff.uni-marburg.de (P.J. Behrendt),
hampp@staff.uni-marburg.de (N. Hampp).
1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2012.08.012

D. Kehrloesser et al. / Journal of Photochemistry and Photobiology A: Chemistry 248 (2012) 8–14
2. Experimental
9
2.1. Materials and methods
All chemicals used were purchased from Sigma–Aldrich (precur-
sors for 2 and 3), Acros Scientific, Merck (TLC plates), ABCR (PPH3),
Fluorochem (TBS-Cl), TCI Europe (DIAD), or Melrob (MAA) and
were used as received. Anhydrous solvents were purchased from
Sigma–Aldrich or Acros Scientific and used as received. All reactions
were done under Schlenk-conditions with argon 4.8 as protective
gas purchased from Air Liquide. The reactions were monitored and
characterized by thin layer chromatography (Merck, TLC silica gel
60 F254 aluminum sheets), 300 MHz 1H NMR, 282 MHz 19F NMR
and 75 MHz 13C NMR spectra (Bruker, Advance 300 spectrometer).
Single photon absorption in solution was induced with a Shimadzu
RF-1502 spectrometer using an excitation wavelength of 266 nm.
The light energy exposed to the samples was determined by using
azobenzene as an actinometer following Gauglitz and Hubig [31].
Irradiation at 532 nm was done utilizing an Infinity 40–100 (Coher-
ent Inc.), a q-switched Nd:YAG laser with a pulse length of 3 ns
and a repetition rate of 40 Hz. The beam has a flat top profile and
the diameter was adjusted to 5 mm by an aperture. The energy at
the location of the sample was measured using a Fieldmaster GS
(Coherent) set to the appropriate wavelength and equipped with a
power meter head Model 80 (Coherent). Analytical HPLC was per-
formed on a Dionex Ultimate 3000 equipped with a Nucleosil RP18
(3 ␮m Bischoff) column utilizing mixtures of acetonitrile (ACN)
(Rotisolv HPLC grade; Roth) and deionized water (purified by a
TKA-Micro ultra-pure water purification system (0.06 ␮S) and acid-
ified with 300 ␮l l−1 phosphoric acid) as eluent. Peak detection was
enabled by diode array detector (DAD). Quantitative measurements
were done by peak integration and referred to calibration standards
of the compounds examined. All UV–vis-spectra were recorded
on a Lambda 35 spectrometer (Perkin Elmer) between 190 nm
and 700 nm with 480 nm min−1 in steps of 1.0 nm. Measure-
ments in solution were performed in quartz cuvettes (QS, Hellma;
UQ, Portman Instruments) with 10 mm path length in the given
solvents.
Fig. 1. General mechanism of the photolysis of ortho-nitrobenzyl compounds:
X = released compound, Y1 = additional nitro group, Y2 = electron donating group
(e.g. OMe), OR = possible linkage (e.g. to polymer).
drug from the polymer and an undesired decomposition of the
UV-absorber. Further the UV absorber may diminish or even
suppress the two-photon controlled release of drug from the
polymer.
We addressed this problem and found that a suitably selected
UV absorber is nicely compatible with the two-photon absorption
triggered drug release from the polymer. As a testbed we used an
ortho-nitrobenzyl group as a linker, 5-fluorouracil as a model drug,
and 2(4-benzoyl-3-hydroxy phenoxy) ethyl acrylate (BHP-EA) as
an UV absorber. 5-FU was chosen, because it is able to address the
secondary cataract inducing proliferating epithelia cells and its low
therapeutical dosage enables the storage of enough drug within
a single IOL. We have prepared IOLs from this newly synthesized
polymer and analyzed the drug release function under two-photon
excitation.
Only very few publications deal with TPA of ortho-nitrobenzyl
compounds. In 2003 Diaspro et al. investigated the two-photon
induced properties of 2-nitrobenzaldehyde as a caged proton
compound utilizing fluorescent labeled nanocapsules as a new sen-
sor [27]. Three years later Aujard et al. published the uncaging
cross-sections for one and two-photon excitation of ortho-
nitrobenzyl based protecting groups with red-shifted absorptions
[28]. Recently Banerjee et al. reported a multifunctional magnetic
nanocarrier which enables the release of a drug conjugated by an
ortho-nitrobenzyl moiety by absorption of near infrared irradia-
tion due to up-conversion of the irradiated energy [29]. Two years
ago Agasti et al. reported the photochemical drug release of 5FU
from gold nanoparticles utilizing a nitrobenzyl moiety similar to
the synthesized monomer in this work [30]. However such mate-
rials are intensely colored and may not be used in applications like
intraocular lenses.
2.2. Synthetic procedures
3-Hydroxy-4-methoxy-2,6-dinitrobenzaldehyde (2) and 3-
hydroxy-4-methoxy-2,6-dinitrobenzyl alcohol (3) as well as
5-iodo-1-tert-butyldimethylsilyloxypentan and 1-N-benzoyl-5-
fluorouracil (Bz5FU) were synthesized following literature proce-
dures [30,32–34].
2.2.1. 3-(5-(Tert-butyldimethylsilyloxy)pentyloxy)-4-methoxy-
2,6-dinitrobenzylalcohol (4)
5 mmol of 3 and 25 mmol of potassium carbonate were sus-
pended in 50 ml of N,N-dimethylformamide (DMF). After addition
of 5.5 mmol of the alcylhalide the suspension was heated to 70 ◦C
for 48 h. After cooling to room temperature (RT) all insoluble salts
were filtered off. The reaction solution was diluted with 150 ml
of deionized water and extracted 3-times with ethyl acetate. The
combined organic layers were washed with brine and dried over
anhydrous sodium sulfate. Evaporation of the solvent yielded the
crude product which was further purified by column chromatogra-
phy on silica gel with pentane and ethyl acetate (4:1) as eluent.
1.26 g, 2.9 mmol, 57% yield: 1H NMR result (300 MHz, CDCl3):
ı/ppm = 7.70 (s, 1H, CarH), 4.70 (d, 2H, 3J = 7.3 Hz, Car CH2 OH),
4.25 (t, 2H, 3J = 6.6 Hz, Car
(t, 2H, 3J = 6.3 Hz, CH2–OTBS), 1.79–1.69 (m, 2H, CH2 aliphatic),
O CH2 CH2), 3.99 (s, 3H, O CH3), 3.62
To our knowledge, this is the first report on two-photon absorp-
tion induced drug release in presence of an UV-absorber from an
optically clear and more or less colorless polymer using an ortho-
nitrobenzyl moiety as the photo cleavable linker where the drug is
released without any auxiliary groups attached.
1.61–1.51 (m, 4H, CH2 aliphatic), 0.89 (s, 9H, Si
C (CH3)3), 0.05 (s,
6H, Si (CH3)2). 13C NMR result (75 MHz, CDCl3): ı/ppm = 152.46,
144.62, 121.27, 110.28, 75.48, 62.92, 57.07, 56.84, 32.32, 29.61,

10
D. Kehrloesser et al. / Journal of Photochemistry and Photobiology A: Chemistry 248 (2012) 8–14
25.94, 25.62, 21.83, 18.33, 1.00, −5.31. HRMS (ESI): m/z = 445.2008
[M+H+] ꢀmmu = 0.7
2.2.2. 3-N-(3-(5-(Tert-butyldimethylsilyloxy)pentyloxy)-4-
methoxy-2,6-dinitrobenzyl)-1-N-benzoyl-5-fluoro-uracil 5
At 0 ◦C 14 mol of diisopropyl azodicarboxylate (DIAD) was added
slowly to a suspension of 14 mmol of triphenylphosphine (PPh3)
in 40 ml anhydrous tetrahydrofuran (THF) and stirred for addi-
tional 30 min. 20 ml (7 mmol) of this preformed beige complex
suspension was added slowly to a suspension of 7 mmol Bz5FU
and 7 mol of 4 in 20 ml anhydrous THF at −20 ◦C. The reaction
mixture was stirred for 15 h and allowed to warm to RT. After
evaporation of the solvent the crude reaction mixture was puri-
fied by column chromatography on silica gel with pentane and
tert-butylmethylether (1:1) as eluent. 3.7 g, 5.6 mmol, 80% yield:
1H NMR result (300 MHz, CDCl3): ı/ppm = 7.95 (d, 2H, 3J = 7.3 Hz,
Bz: Cq CH CH) 7.70–7.65 (m, 2H, CarH
C NO2, Bz: Cq CH CH),
7.52 (t, 2H, 3J = 7.3 Hz, Bz: CH CH CH), 7.28–7.26 (m, CH CF,
solvent signal), 5.07 (s, 2H, Car CH2 N), 4.29 (t, 2H, 3J = 6.6 Hz,
Car
CH2–OTBS), 1.81–1.72 (m, 2H, CH2 aliphatic), 1.61–1.51 (m, 4H,
CH2 aliphatic), 0.90 (s, 9H, Si (CH3)3), 0.06 (s, 6H, Si (CH3)2).
O
CH2 CH2), 4.02 (s, 3H, O CH3), 3.63 (t, 2H, 3J = 6.3 Hz,
C
19F NMR result (282 MHz, CDCl3): ı/ppm = −163.4. 13C NMR result
(75 MHz, CDCl3): ı/ppm = 166.62, 153.57, 148.13, 144.86, 144.26,
138.53, 135.45, 130.75, 130.51, 129.25, 127.22, 126.77, 113.87,
110.58, 75.79, 62.88, 57.02, 44.24, 32.31, 32.10, 29.61, 28.60,
25.94, 22.78, 21.88, 18.34, 1.00, −5.30. HRMS (ESI): m/z = 683.2155
[M+Na+] ꢀmmu = −0.1
Fig. 2. Synthesis of 5FU loaded photoactive monomer.
aldehyde with sodium borohydride [30] tert-butyldimethyl pro-
tected pentanol was introduced at the more reactive phenolic
alcohol in 3-position utilizing Williamson’ ether synthesis. In the
second step 5FU was coupled to the precursor (4) using the Mit-
sonobu reaction to form the desired amine bond. This synthesis
was adopted from the former reported Mitsonobu coupling of N-3-
benzoylthymine with various alcohols [35] and increased the yield
of the deprotected drug loaded precursor from 5% over four steps
[30] to 40% over three steps and lowered the reaction time from 73 h
to 33 h. Finally 5 was deprotected and esterified with methacrylic
acid to yield a monomer suitable for photo chemical drug delivery
(6) which was copolymerized with a MMA/HEMA mixture (Fig. 2).
2.2.3. 3-N-(3-(5-Methacryloxypentyloxy)-4-methoxy-2,6-
dinitrobenzyl)-5-fluorouracil 6
To 3 mmol of 5 dissolved in 15 ml methanol 15 ml of concen-
trated hydrochloric acid was added. The resulting solution was
stirred at 60 ◦C for 15 h. After cooling to RT the reaction mixture was
diluted with 50 ml of deionized water and extracted with chloro-
form for three times. The combined organic layers were dried over
anhydrous sodium sulfate and the solvent was evaporated under
reduced pressure. The sustained crude product was purified by col-
umn chromatography on silica gel with chloroform and methanol
(15:1) as eluent. At 0 ◦C 2.75 mmol (556 mg) of DIAD was added
slowly to a suspension of 2.75 mmol (710 mg) of PPh3 in anhydrous
THF and stirred for additional 30 min. This preformed beige com-
plex suspension was added slowly to a suspension of 2.75 mmol
(233.2 ␮l) of methacrylic acid (MAA) and 2.5 mmol of deprotected
5 in anhydrous THF at −20 ◦C. The reaction mixture was stirred for
3 h and allowed to warm to RT. After evaporation of the solvent the
crude reaction mixture was purified by column chromatography
on silica gel with pentane and ethyl acetate (3:2) as eluent result-
ing in 702 mg (1.375 mmol, 55% yield) of white solid. H NMR result
(300 MHz, CDCl3): ı/ppm = 7.70 (s, 1H, CarH), 7.18 (d, 1H, J = 5.7 Hz,
CH CF), 6.11 (m, 1H, CH2 Cq), 5.56 (p, 1H, J = 1.5 Hz, CH2 Cq), 5.04
3.2. Photochemical characterization—selection of UV-absorber
Fig. 3 shows the absorption spectra of 4, 5 and 6 as well
as the spectrum of the UV-absorber BHP-EA at a concentra-
tion of 0.03 mM. The ortho-nitrobenzyl precursor 4 has a weak
broad absorption between 260 nm and 400 nm with a maximum
at 324 nm. After functionalization with benzoyl-5-fluorouracil
(Bz5FU) (5), a stronger absorption band with a maximum at 252 nm
(s, 2H, Car CH2 N), 4.28 (t, 2H, 3J = 6.4 Hz, Car
O CH2 CH2), 4.16
(t, 2H, 3J = 6.5 Hz, CH2
O
(C O)), 4.02 (s, 3H, O CH3), 1.94 (m, 3H,
CH2 Cq CH3), 1.82–1.68 (m, 4H, CH2 aliphatic), 1.53–1.46 (m, 2H,
CH2 aliphatic). 19F NMR result (282 MHz, CDCl3): ı/ppm = −164.8.
13C NMR result (75 MHz, CDCl3): ı/ppm = 153.49, 148.95, 146.82,
144.60, 144.45, 138.86, 136.41, 129.53, 128.33, 127.65, 127.20,
125.33, 113.96, 110.51, 75.42, 64.35, 57.03, 43.97, 29.41, 28.20,
22.09, 18.28. HRMS (ESI): m/z = 533.1287 [M+Na+] ꢀmmu = −0.3.
3. Results and discussion
3.1. Synthesis of drug loaded monomer
Synthesis started from isovanilin, which was nitrated in 2-
and 6-position by concentrated nitric acid. After reduction of the
Fig. 3. Absorption spectra of 0.03 mM solutions of 4, 5 and 6 in ACN.

D. Kehrloesser et al. / Journal of Photochemistry and Photobiology A: Chemistry 248 (2012) 8–14
11
Fig. 5. Determination of quantum efficiency from the change in concentration
versus the absorbed energy for 5.
a 1 mM solution of 5 after consecutive irradiations at 266 nm. The
increasing signal at a retention time of 1.8 min corresponds to the
released Bz5FU, the decreasing signal at 10.7 min correspond to
cleaved 5 and the nitroso-aldehyde generated during the photo
induced release process. They have the same retention time but can
be identified due to their different absorption spectra. To quantify
the degree of cleavage the peak area of Bz5FU was recorded and
correlated to Bz5FU standards of known concentration.
Fig. 4b depicts the disassembly of the caging compound in
the spectral range of 190–250 nm. The range 250–305 nm stands
for the buildup of secluded drug molecules, whereas the region
beyond that represents the formation of the nitroso-compound.
The isosbestic points at 250, 280 and 305 nm, together with the tidy
chromatogram from Fig. 4a stand for a clean uncaging reaction.
The efficiency of the photo cleavage at low conversion in the
beginning of the reaction is quantified by the single photon quan-
tum yield ϕcleave
Fig. 4. (a) HPLC chromatograms of a 1 mM solution of 5 after consecutive irradi-
ations with 266 nm light with total energies given. (b) Change in absorption after
irradiation with 266 nm (total energies given) of a 25 ␮M solution of 5 in ACN.
is observed. Deprotection of 5FU and esterification with MAA shifts
the absorption band of the monomer 6 to 268 nm which corre-
sponds well to the main absorption of the unfunctionalized free
drug. Irradiation with 266 nm, i.e. single photon absorption, or with
532 nm for the TPA process results in excitation of the molecule
near the maximum of the absorption of 5FU. It is likely that the
majority of the energy gets absorbed by the uracil part and then
to be transferred to the nitrobenzyl moiety. Longer lifetimes for
nucleobase-compounds have been measured by Kohler et al., which
would make such an energy transfer supposable [36]. Steiner et al.
have investigated comparable substances and could show that the
energy transfer from a linked chromophor to the nitrobenzyl part
was around double figure ns. But conclusively the H-atom transfer
of the uncaging mechanism seems to determine the lifetime of such
combined chromophors [37,38]. A more detailed analysis can only
come from transient absorption measurements. But the absorption
of 6 corresponds quite nicely with the chosen UV-absorber BHP-AE,
which is an efficent UV-blocker in the range between 275 nm and
350 nm. At a wavelength of 266 nm it exhibits a minimum in the
spectrum preserving the possiblity of TPA induced drug release,
assuming that BHP-AE remains uneffected by the irradiated pulsed
laser light. This was checked by irradiation of 3 mM solution of
BHP-AE with 25.5 kJ of laser light at 532 nm. Neither in the HPLC
chromatogram nor in the UV–vis spectra of the irraditated solution
any change in retention time or absorbance was monitored.
ndrug
ndrug
Eabs
ϕcleave
=
=
·
(I)
nphoton
Eabs nphoton
ndrug represents the number of cleaved molecules, nphoton the num-
ber of photons absorbed and Eabs the absorbed energy. The number
of cleaved molecules per absorbed energy can be calculated by
ndrug
Eabs
cdrug
Eabs
=
· V · NA
(II)
with cdrug being the concentration of cleaved molecules, V the vol-
ume of the probe (1 ml) and NA the Avogadro constant. Plotting the
concentration of cleaved dimers versus the actual energy absorbed
(Fig. 5) results in a linear increase with cdrug · Ea−b1s as the slope of the
linear fitting of the data points. With (II) ndrug · E−1 is calculated to
abs
be 3.64 × 1017
J
−1. The number of photons per energy unit results
in 1.34 × 1018 J−1 according to (III)
nphoton
ꢁ
=
(III)
Eabs
c · h
ꢁ represents the wavelength, c the speed of light and h the Planck
constant. Using (I) the quantum yield of the cleavage for 5 is deter-
mined to be 27% corresponding well with the literature [39].
3.4. Two-photon induced drug release in solution
3.3. Single photon induced drug release in solution
To verify that the drug release is possible in a two-photon
absorption triggered process a 0.2 mM solution of 5 in ACN was
irradiated with 532 nm at different pulse energies over a constant
time. Fig. 6a shows the change in absorption at 370 nm, which
is proportional to the drug release versus the irradiation time at
In order to avoid undesired side reactions the protected
monomer precursor 5, which has the same chromophor structure
as the reactive monomer 6, was characterized in its photochem-
ical properties in solution. Fig. 4a shows HPLC chromatograms of

12
D. Kehrloesser et al. / Journal of Photochemistry and Photobiology A: Chemistry 248 (2012) 8–14
Fig. 7. (a) HPLC chromatograms of a 6.05 mM solution of 5 after consecutive irra-
diations with 532 nm light pulses with the total energies given. (b) Change in
concentration of released Bz5FU over irradiation time.
Fig. 6. (a) Absorption at 370 nm versus time of irradiation at different pulse energies
and (b) double logarithmic plot of reaction rate over pulse energy.
different pulse energies. Because the efficacy of the TPA-induced
excitation depends on the square of the light intensity, the drug
release is expected to be proportional to the square of the applied
laser intensity. Fig. 6b shows a double logarithmic plot with a slope
of 1.66 indicating a TPA process. The deviation from a slope value of
2 could arise from non-linear absorption effects from the free drug
molecule.
To quantify the TPA mediated drug release a 6.05 mM solution
of 5 in ACN was irradiated at 532 nm with increasing energy doses.
The photochemical release process was monitored by HPLC.
Again the increasing signal at a retention time of 1.8 min in
Fig. 7a corresponds to the released Bz5FU, the decreasing signal
at 10.7 min corresponds to cleaved 5 and the nitroso aldehyde gen-
erated during the photo-induced release process. The TPA cross
section ꢂTPA can be calculated from Eq. (IV)
For this purpose a mixture of 0.4 wt% (6.05 mM) of 5 and 0.1 wt%
(3.2 mM) of BHP-EA corresponding to a 1 mm thick IOL with the
common amount of 1 wt% of UV-absorber and a active drug load of
1 wt%, was irradiated in a 10 mm cuvette with pulsed laser light at
532 nm. Fig. 8a shows the HPLC chromatograms of the irradiated
solution, as well as the amount of released Bz5FU over the irradiated
energy compared to the achieved release from a solution without
BHP-EA. The signal at a retention time of 3.2 min corresponds to
BHP-EA and remains unaffected over the complete irradiation. As
Fig. 8b shows the presence of BHP-AE lowers the total amount of
released Bz5FU by 6% only.
3.5. Two-photon induced drug release from polymer
A mixture of 86.5 wt% HEMA, 12 wt% MMA, 1 wt% EGDMA as
cross linker and a combination of 0.25 wt% of campherchinone and
0.25 wt% of ethyl-4-aminobenzoate as photo initiator at 465 nm
served as basic monomer solution. Addition of 1 wt% of BHP-EA
and 4 wt% of 6 to this solution resulted in the final polymeriza-
tion mixture, corresponding to a net drug load of 1 wt% of 5FU. The
resulting slightly yellow transparent plate (Fig. 9a) was extracted
with distilled water over 3 days to remove the initiator and unre-
acted monomer. A maximum water uptake of about 22.8% was
reached. TPA induced drug release from the polymer matrix could
be achieved in two steps by irradiation of the polymer plate with
0.85 kJ each at 532 nm. The UV absorption at 268 nm of the sur-
rounding water the polymer plate was immersed in, was monitored
and the diffusion time and the final amount of released drug were
determined (Fig. 9b). The diffusion time from the model polymer
plate was very fast. A steady state 5FU concentration was reached
within 10 min after the irradiation. The drug release results in an
increasing absorption at 370 nm (for comparison see Fig. 4b). This
ꢀ
ꢁ
2
dc
dt
h · c · A · t
E · ꢁ
1
ꢂTPA
=
· Vtotal
·
·
(IV)
˚
TPA · c0 · Vir
With dc/dt corresponding to the change in concentration of released
Bz5FU over time (Fig. 7b), Vtotal to the total sample volume (1.5 ml),
E to the pulse energy, A to the irradiated area (0.196 cm2), t to the
pulse length (3 ns) ˚TPA to the TPA quantum yield, c0 to the starting
concentration of the irradiated solution and Vir to the irradiated
volume (0.196 cm3). Under the assumption that the quantum yield
of the TPA is equal to the SPA quantum yield a TPA cross section of
2.41 GM can be obtained. This value exceeds the measured values
of other o-NBnCs in the range of 0.01–0.1 GM [28] by more than
one order of magnitude. This value is about the same compared to
the TPA cross sections of photochemical drug delivery alternatives
like coumarin or tetralone homo or hetero dimers [19,21].
The next step was to prove that the TPA triggered drug release is
still possible in the presence of BHP-EA as the UV absorbing agent.

D. Kehrloesser et al. / Journal of Photochemistry and Photobiology A: Chemistry 248 (2012) 8–14
13
Fig. 8. (a) HPLC chromatograms of a 6.05 mM solution of 5 in the presence of BHP-
EA after consecutive irradiations with 532 nm light with the total energies given. (b)
Amount released Bz5FU with and without BHP-AE.
Fig. 10. (a) Prototype IOL in front of a human eye. (b) Absorption spectra of 5FU
released by TPA from the IOL.
absorption band, corresponding to the generated nitroso function-
ality, unfortunately causes a slight increase in yellow color of the
polymer plate at the point of irradiation (Fig. 9b).
As a final proof-of-principle model IOLs having a ‘Saturn-ring’
haptic were fabricated. The IOLs were prepared from polymer
plates by CNC diamond turning, polishing and extraction with phys-
iological saline solution. Finally the IOLs were packaged and steam
vapor sterilized. The diameter of the whole IOLs was 7.5 mm, the
diameter of the lens was 5.0 mm having a thickness of 0.9 mm. The
optical power was determined to be 22.5 dpt (Fig. 10a).
Finally the release of unmodified 5FU by TPA excitation at
532 nm in therapeutical relevant amounts of about 0.5–1.0 ␮g per
dose from an UV-absorber containing foldable hydrophilic IOL was
demonstrated. The drug diffused out into the aqueous environment
within minutes. The IOL was irradiated two times with 0.65 kJ at
532 nm in 1.5 ml of physiological saline solution. The absorption at
268 nm in Fig. 10b corresponds to the released 5FU. After irradiation
with 1.3 kJ the concentration of released 5FU reached 1.5 ␮g ml−1
,
proving that the estimated therapeutical dosage of 1.0 ␮g ml−1 was
reached easily.
4. Summary and conclusion
We demonstrated that two-photon triggered drug release from
polymers can easily be accomplished in the presence of an UV-
absorber. The UV absorber should be selected to have a minimum
of absorption at the wavelength used for excitation of the molecu-
lar moiety excited to uncage the drug. In this manuscript we used
a new model compound with an ortho-nitrobenzyl-5-fluorouracil
Fig. 9. (a) Drug loaded polymer plate before (left) and after irradiation at 532 nm
with 0.85 kJ. (Right) (b) concentration of released 5FU from the polymer from two
consecutive irradiation steps. (For interpretation of the references to color in text,
the reader is referred to the web version of this article.)

14
D. Kehrloesser et al. / Journal of Photochemistry and Photobiology A: Chemistry 248 (2012) 8–14
functionality for single-photon and two-photon drug release. The
quantum yield for SPA induced drug delivery could be calculated to
0.27 and the TPA cross section at 532 nm was found to be 2.41 GM. In
presence of the BHP-EA no significant decrease of the TPA induced
drug release in solution was measured.
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