Photosensitizer drug delivery via an optical fiber

Article abstract of DOI:10.1021/ja200840p

An optical fiber has been developed with a maneuverable mini-probe tip that sparges O₂ gas and photodetaches pheophorbide (sensitizer) molecules. Singlet oxygen is produced at the probe tip surface which reacts with an alkene spacer group relea

Full text of DOI:10.1021/ja200840p

ARTICLE
pubs.acs.org/JACS
Photosensitizer Drug Delivery via an Optical Fiber
Matibur Zamadar, Goutam Ghosh, Adaickapillai Mahendran, Mihaela Minnis, Bonnie I. Kruft,
Ashwini Ghogare, David Aebisher, and Alexander Greer*
Department of Chemistry and Graduate Center, City University of New York, Brooklyn College, Brooklyn, New York 11210,
United States
S Supporting Information
b
ABSTRACT: An optical fiber has been developed with a maneuverable mini-
probe tip that sparges O2 gas and photodetaches pheophorbide (sensitizer)
molecules. Singlet oxygen is produced at the probe tip surface which reacts with
an alkene spacer group releasing sensitizer upon fragmentation of a dioxetane
intermediate. Optimal sensitizer photorelease occurred when the probe tip was
loaded with 60 nmol sensitizer, where crowding of the pheophorbide molecules
and self-quenching were kept to a minimum. The fiber optic tip delivered
pheophorbide molecules and singlet oxygen to discrete locations. The 60 nmol
sensitizer was delivered into petrolatum; however, sensitizer release was less
efficient in toluene-d8 (3.6 nmol) where most had remained adsorbed on the
probe tip, even after the covalent alkene spacer bond had been broken. The results open the door to a new area of fiber optic-guided
sensitizer delivery for the potential photodynamic therapy of hypoxic structures requiring cytotoxic control.
’ INTRODUCTION
scission of a dioxetane intermediate.11 Thus, dioxetane cleavage
could be conducted for sensitizer release to effectively increase the
short diffusion distance of 1O2 away from the probe tip
Current photodynamic therapy (PDT) methods all employ
systemic administration of dyes.1 Instead of injecting a photo-
sensitizer into a patient with irradiation, the PDT of tumors could
benefit from a fiber optic that guides the photosensitizer to a
specific location. However, at present, no point-source fiber-
optic 1O2 generator exists as an alternative PDT method.
The study reported here describes the efficacy of sensitizers to
cleave free from the porous silica caps of the fiber optic in
Figure 1. The probe tip photocleaves pheophorbide formate
ester (3) and leaves behind cofragment 4. Hydrolysis products
include 4-hydroxybenzylic alcohol and formic acid, where the
amount of formic acid generated is far less than what is irritating
(13 mmol, open skin) or lethal (LD50 in mice is 700 mg/kg).2
Our system uses visible light to cleave the photosensitizer
away from a solid surface. Photocleavable groups on solids or
biological surfaces typically use UV light, such as the benzoin,3
nitrobenzene,4 phenyacyl,5 and coumarin systems,6 although
some have used 2-photon excitation.7 The previous work of
Dolphin8 and Breslow9 employed visible light in solution phase
photocleaving and drug-delivery systems.
1
(∼150 nm in water and less in vivo12). The previous O2 fiber
optic we developed lacks a potential PDT utility due to the
1
extremely short diffusion distance of O2 away from the probe
tip13 in the absence of a photocleavable sensitizer.
The manuscript contains three parts: (1) the synthesis of the
new heterogeneous sensitizer 1, (2) the optimal loading of
sensitizer for maximum visible-light photorelease, and (3) the
percent disappearance of starting material and the percent yield
of surface-released sensitizer in different media. Our aim was to
open the door to a brand new area of fiber-optic guided drug
delivery whose origins come from concepts in organic synthesis
and molecular photochemistry. Unexpectedly, the probe tip had
adsorbtive affinity for sensitizer 3 in toluene, while its quantita-
tive departure took place in the semisolid petrolatum. Sensitizer
3 localization into the petrolatum indicates a potential for local
dye delivery via permeability enhancement.14
’ RESULTS AND DISCUSSION
Our hypothesis was that sensitizer molecules will cleave away
from the fiber optic probe tip in a “three-phase” experiment: the
gas phase was the hollow core of the fiber, the solid phase was the
fiber cap, and the “outer” phase was the bulk solution or
semisolid media. O2 gas flowed from a compressed oxygen tank
to a T-valve in the custom optical fiber, which was connected to
the sensitizer cap 1 via a Teflon inner flow tube. A pheophorbide
derivative was selected as the 1O2 sensitizer,10 and a Z-enol ether
was selected as the spacer group bridging the sensitizer and the
glass tip, which can react with 1O2 and be cleaved apart by way of
1. Synthesis of the Heterogeneous Sensitizer. We sought a
versatile heterogeneous sensitizer for rapid photorelease, and
designed the pheophorbide/alkene conjugate as a new photo-
cleavable sensitizer. The conversion of 4-bromophenol (5) to cis-
1,2-bis(4-bromophenoxy)ethene (8) was carried out in 3 steps
using a known procedure (Scheme 1).15 Meso-7 and dl-7 were
Received: January 27, 2011
Published: May 03, 2011
r
2011 American Chemical Society
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|

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Figure 1. Concept of the singlet oxygen fiber optic: (1) illuminator-to-fiber coupling, (2) compressed oxygen-to-fiber coupling via a flare T-valve to a
borosilicate fiber optic consisting of a Teflon gas flow tube, (3) porous Vycor glass (PVG) cap-to-fiber coupling, (4) photocleavable sensitizer solid, (5)
internally flowing light and oxygen, externally produced 1O2, [2 þ 2] cycloaddition at the alkene site, (6) cleavage of sensitizer 3 free from the probe tip
via the scission of dioxetane 2, and (7) production of cofragment 4 and hydrolysis byproduct.
formed, and a separation of the meso was necessary to reach 8,
which reacted with n-BuLi and DMF to generate the bis-aldehyde
(9) in 60% yield. Bis-aldehyde 9 reacted with sodium borohy-
dride quantitatively to give “spacer group” 10. Spacer group 10
was prepared as a photocleavable group bridging the silica cap
and the photosensitizer. Pyropheophorbide-a reacted with 10,
EDC, and DMAP yielding pyropheophorbide monoester (11),
which was purified and isolated in 60% yield. Sensitizer silane 12
and 3-iodopropyltrimethoxy silane were then covalently bonded
to the porous Vycor glass (PVG) (step vii, Scheme 1), which
consisted of free, isolated or associated, and hydrogen bonded
clusters of silanol groups, SiꢀOH.16,17 In 1, the ratio of the
sensitizer-to-iodosilane-to-silanol sites was typically ∼1:3:300,
but as will be seen in Section 2, tunable loading amounts were
critical for optimal and controllable sensitizer release. Solid 1 was
stable in the dark, no sensitizer leaching was observed when the
material was (i) repeatedly washed with toluene, THF, chloro-
form, ether, and hexane solvents; (ii) Soxhlet extracted with
chloroform and ethanol; or (iii) immersed in water solution at
pH 4ꢀ7 for 4 h at room temperature. Because the filtrates of
(i)ꢀ(iii) showed no photosensitizer activity, we concluded that 1
contained siloxane bonds where the sensitizer was chemically
bound to the silica matrix. The FTIR data further bolstered the
structural assignment of the saturated carbons of the spacer
methylene groups of 1 (Figure 2). The depth that the sensitizer
penetrated into PVG was examined using a microscope equipped
with a CCD camera. Figure 3 shows a sensitizer-attached PVG
sample 1, cut so that the ∼0.08 mm depth and localization of the
sensitizer on the outer face of the cap could be viewed. Unlike
conventional gasꢀliquid systems that introduce O2 gas into the
liquid phase,18 the present system is improved and transmits O2
gas through the pores of the PVG membrane tip where an
anaerobic solution becomes oxygen saturated with access to the
covalently excited sensitizer sites (Figure 4). The next step was
determining the optimal loading of sensitizer for maximum
photorelease from the solid surface.
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Scheme 1. Synthesis of Sensitizer Functionalized Cap 1a
a Reagents and conditions: (i) BrCH2CH2Br, NaOH, 100 °C, 6 h; (ii) NBS, benzoyl peroxide, CCl4, 80 °C, 6 h (meso-7 was carried on to step iii); (iii)
NaI, acetone, 25 °C, 2 h; (iv) n-BuLi, DMF, THF, ꢀ78 °C, 3.5 h; (v) NaBH4, CH3OH, 25 °C, 14 h; (vi) pyropheophorbide-a, EDC, DMAP, CH2Cl2,
25 °C, 24 h; (vii) (CH3O)3SiCH2CH2CH2I, NaH, THF, under N2, 70 °C, 24 h; (viii) porous Vycor glass (predried at 500 °C), toluene, reflux 110 °C, 24 h.
Figure 3. A low-magnification (10ꢁ ) cross-sectional optical image.
The dark green thin coating shows the depth of 1 accessed into PVG.
The image shows ∼0.08 mm penetration depth on the outer face of the
cylinder-shaped PVG cap 1 (diameter 5.0 mm ꢁ length 8.0 mm).
sensitizer molecules can be estimated with eqs 1ꢀ4
(Experimental Section), in which a simple surface geometry
Figure 2. The FTIR spectrum of functionalized porous glass 1, in which
the CꢀHx stretching modes observed at 2851 and 2954 cmꢀ1 were
was assumed. The loading of 0.3-μmol (0.33%) sensitizer onto
the fiber caps resulted in maximal photocleavage of 3. Higher or
lower sensitizer loading reduced the photocleavage efficiency and
was attributed to less available sensitizer and self-quenching,
respectively (Figure 5). For example, a 3-fold mole increased
loading of 12 resulted in an 11-fold decrease in sensitizer
photocleavage (cf. entries 2 and 5, Table 1). This suggests that
dye molecules sufficiently isolated from each other (>∼17 nm)
efficiently cleave with minimal dipoleꢀdipole energy transfer
due to congestion (F€orster pheophorbide radius = 6.2 Å).19 The
stability of photocleaved 3 in methanolꢀwater solution (9:1) at
assigned to saturated carbons of the spacer methylene groups indicating
that 12 and (CH3O)3SiCH2CH2CH2I (1:3 ratio) were anchored to the
PVG surface. The FTIR spectrum of a clean piece of PVG was not
identical, no CꢀHx stretching modes were observed.
2. Optimal Loading of Sensitizer for Maximum Photore-
lease. Silane 12 quantities of 0.06ꢀ1.1 μmol were loaded onto
PVG per gram resulting in sensitizer sites separated by
8.9ꢀ38.4 nm (Table 1). A likely spatial distance between the
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Figure 5. Percent of 3 photoreleased into toluene-d8 solution from the
porous cap 1. Silane 12 was loaded in 0.06ꢀ1.1 μmol amounts
(0.068ꢀ1.24%) onto porous Vycor glass per gram and was exposed to
white light.
sensitizer 3 remained adsorbed on the probe tip even though the
alkene bond bridging the sensitizer and glass was broken (trace c,
Figure 7). The sensitizer photorelease chemistry was worse in
D2O, as indicated by the observation that 3 was not detected in
D2O solution (entry 8, Table 2). An effort to separate 3 from the
glass surface was successful with Soxhlet extraction, where
photolyzed 1 resulted in the dissociation of the nonpolar 3
adsorbate into the surrounding solution.
Figure 4. Schematic of the sensitizer functionalized porous cap 1 where
oxygen and light come internally from the hollow optical fiber. Typically
0.3 μmol or 0.33% silane 12 was loaded per gram of PVG. The sensitizer
may adopt various conformations on the isotropic PVG material.
Table 1. Loading of Pheophorbide Silane 12 onto
Porous Vycor Glass (PVG)a
An interesting result was this in contrast to toluene-d8 and
D2O, the sensitizer payload release was efficient in petrolatum
(soft paraffin, mixture of hydrocarbons) at 65 °C. We have
considered petrolatum as an adequate semisolid medium, that is,
for physical organic studies with relevance to lipophilic biological
media. After 30 min, the diffusion distance of the sensitizer away
from the fiber-optic tip was 1.03 mm, and the geometry of the
spot was approximately circular (Figure 8). After 4 h, quantitative
sensitizer departure occurred in petrolatum. No significant
adsorption of the sensitizer occurred with the petrolatum, the
sensitizer remained in the surrounding semisolid phase (trace d,
Figure 7). Thus, it is evident that the surrounding medium
influences the photorelease efficiency at the probe tip. By
monitoring the course of the reaction, the progress of the
photorelease could be scrutinized. With the fiber optic delivering
visible light and oxygen to the sensitizer tip, a rapid 47%
photodegradation of the alkene bonds was observed in 30 min
(trace a, Figure 7). After about 2 h, 92% of the alkene bonds were
cleaved, whereas full photodegradation of the alkene bonds
required 4 h to complete. Because the covalently attached
sensitizer reached a 0.08 mm depth, the observed fast and slow
alkene photodegradation components can be attributed to the
depth in which the sensitizer is linked and the geometric isolation
of the latter.
Indirect evidence for the intermediacy of a surface-bound 1,2-
dioxetane species shown in Scheme 3 is also reported. Singlet
oxygen reacted with the alkene bond of glass 1 (0.6 g) producing
dioxetane 2 which deoxygenated to an epoxide (16), likely via a
phosphorane (15),20 by trimethylphosphite (0.6 M). The
amount of trimethylphosphate detected was 0.57 ( 0.09% of
the number of alkene sites after ∼70% conversion in toluene-d8.
A homogeneous photolysis experiment of diol 10 in toluene-d8
was conducted in a similar manner, but showed a 6-fold increase
in dioxetane trapping than could be achieved with the hetero-
geneous system 2. We propose the sensitivity of dioxetane 2
comes by virtue of its proximity to the silanol and silanoxy anion
surface groups (Scheme 4). Peroxide bond decomposition by
nucleophiles has been discussed before.21 In some cases, anionic
sens/
ave. sensꢀsens
entry loaded, %b SiOH ratio distance (nm)c
photocleavage, %d
1
2
3
4
5
6
0.068
0.33
0.48
0.60
0.99
1.24
1:1470
1:290
1:210
1:170
1:100
1:80
38.4
17.1
14.5
12.9
10.0
8.9
∼2.1
6.06
3.32
1.42
0.53
0.39
a The sensitizer is thinly coated, it reaches a maximum depth of 0.08 mm
into PVG. The percent of sensitizer loaded onto PVG was defined as the
number of sensitizer molecules attached vs the number of silanol groups
at a depth of 0.08 mm. b The sensitizer loadings were varied based on the
ratio of 12 to (CH3O)3SiCH2CH2CH2I added. The range was 0.06ꢀ1.1
μmol sensitizer silane 12 with concomitant decreases of iodosilane from
0.84 to 0.32 μmol. Thus, the 12 to iodosilane ratio ranged from 1:14
to 1:0.3. c Experimental Section describes the calculation of the
sensꢀsens distance. d White light from a Rayonet reactor was used to
photocleave the sensitizer.
pH 2ꢀ8 was also investigated by LCMS. As expected, after
several minutes it remained unchanged, but at high or low pH,
the disappearance of 3 was mostly due to the hydrolysis of the
formate ester bond (path A), and then later there was the
appearance of 14 (path B) (Scheme 2). Each pair of 3 and 4
can potentially liberate 2 equiv of formic acid, for a maximum of
120 nmol formic acid arising from an 0.33% sensitizer-loaded 0.2
g fiber cap, along with 60 nmol 4-hydroxybenzylic alcohol.
3. The Percent Disappearance of Starting Material and the
Percent Yield of Surface-Released Sensitizer in Different
Media. We have carried out a systematic study of the sensitizer
photorelease in different media (Figures 6ꢀ8 Table 2). Figure 6
shows the amount of sensitizer 3, photoreleased into toluene-d8
solution, as a plot of sensitizer release, that is, OD versus time.
The increase in OD indicates the release into solution, which
gave a maximum of 7300 nM of 3. Significant quantities of
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Scheme 2. Stability of Photosensitizer 3 in Acid and Base Solution
Figure 8. Time course of the diffusion of 3 away from the probe tip into
petrolatum at 65 °C.
Figure 6. Time course of photorelease of 3 into toluene-d8 solution
arising from photooxidative cleavage and departure from the fiber optic
device tip. The absorption spectra show the fourth Q-band of 3 and were
normalized at 640 nm: (a) orange 0.0 h, (b) turquoise 0.5 h, (c) blue
1.0 h, (d) green 1.5 h, (e) red 2.0 h, and (f) black 4.0 h.
coming through the tip, 1O2 is located near the surface in a free
or adsorbed state, which reacts with the alkene group with
inefficient diffusion to the surrounding solution. There is a loss
in mobility of 1O2 into the solution phase. Namely, the concen-
1
tration of O2 drops off rapidly in the solution phase with
increasing distance away from the probe tip due to 1O2 uptake
at the alkene sites and the intrinsic short diffusion distance of 1O2.
(iii) Silanol or silanoxy anion nucleophilic attack on a C atom of
the dioxetane group is proposed to take place where the
heterogeneous catalyst causes a gain in the initial yield of 3 from
1 by a mechanism other than higher loading of the sensitizer
silane. Shown in Scheme 6 are two different pathways that can be
visualized for the dioxetane cleavage reaction. The first involves
an adsorption of 3 and the lack of photorelease due to the
resistance to 3 dissolution as revealed by the toluene-d8 and D2O
media at the cap/outer-phase boundary. The second pathway
involves the release of 3, which effectively increases the diffusion
distance of singlet oxygen. In this case, the amount of 3 released
into petrolatum would probably relate to local drug delivery via
permeabilization,23 which is an important finding to distinguish a
new application in PDT. Overall, we ascertained single digit to
tens of nanomoles of sensitizer released, which appears to surpass
the objective of 0.25 nmol sensitizer/mL needed for PDT,24
although the quantities needed can depend on the type of tumor
targeted.25
Figure 7. Reaction profile of fiber optic delivering light and oxygen to
the probe tip (a) percent sensitizer bioconjugate 1, (b) percent surface-
bound dioxetane 2, (c) percent 3 photoreleased into toluene-d8 at room
temperature, and (d) percent 3 photoreleased into petrolatum at 65 °C.
nucleophiles or oxyanion substituents can promote OꢀO bond
homolysis by an electron transfer reaction.22
A reviewer rightfully pointed out that an alternative system
could be designed, where 60 nmol of the sensitizer in an
appropriate solvent is delivered by the same capillary under the
pressure of oxygen without the use of a sensitizer tethered to the
porous tip. Because such pump technology in this fiber optic with
slow oxygen sparging is not yet available, we have not sought this
avenue for the potential facile sensitizer delivery into biological
matrices.
Shown in Schemes 4 and 5 is a proposed mechanism for the
photooxidation and cleavage of the alkene group covalently
bonded to the porous glass surface. The mechanism involves 3
steps: (i) visible light and O2 gas emerge from the opposite face
of the attached sensitizer molecules for a tripletꢀtriplet energy
1
3
transfer reaction producing O2. (ii) Of the O2 molecules
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Table 2. Photorelease of Sensitizer 3a
photocleaved state
entry
medium
irrad. time (h)
free 3 (%)
free 3 (nmol)
free 3 (nM)
comments
1
toluene-d8
toluene-d8
toluene-d8
toluene-d8
toluene-d8
toluene-d8
toluene-d8
D2O
0
0
0.8
2.6
3.7
4.9
6.1
∼70
0
0
0.5
0
960
in dark
a,b,d
2
0.5
1.0
1.5
2.0
4.0
1.5
3.0
0
a,b,d
a,b,d
a,b,d
a,b,d
c
3
1.6
3,110
4,400
5,860
7,300
∼280,000
0
4
2.2
5
2.9
6
3.6
7
∼250
0
a,b,d
8
9
petrolatum
petrolatum
petrolatum
petrolatum
0
0
0
in dark
a,b,e
10
11
12
0.5
1.0
4.0
37
11.1
13.8
30.0
27,750
34,500
75,000
a,b,e
a,b,e
46
100
a Irradiation source: internal irradiation of tip via fiber optic with an output energy density of 444 mJ/cm2. b The 0.2 g PVG cap was loaded with 60 nmol
12 (0.33% surface coverage). PVG fiber tip dimensions: cylinder shape with a length of 8.0 mm, diameter of 5.0 mm, and hole (2.0 length ꢁ 3.0 mm
diameter). c External Rayonet reactor irradiation of 1.0 g PVG loaded with 360 nmol 12 (0.33% surface coverage) in 0.9 mL toluene-d8 followed by
e
Soxhlet extraction to dissociate the adsorbate 3 into the surrounding solution. d Absorption spectroscopy was used for the quantitation of 3. An
epifluorescence microscope was used to detect 3 in petrolatum at 65 °C (0.4 mL).
Scheme 3
Scheme 4
’ CONCLUSION
the dark. The fiber optic was configured so that visible light and
O2 gas were coadministered through the porous fiber optic cap,
and was capable of O2 sparging to hypoxic sites. (2) Sensitizer
loading was optimized, the maximal photocleavage arose when the
surface distance between sensitizers was ∼17 nm. Lower sensitizer
loadings reduced the photocleavage efficiency due to less avail-
able sensitizer. Higher loadings were inefficient likely due to
the crowding of sensitizer molecules and sensitizer-sensitizer
There is now a photosensitizer/fiber optic system usable as a
point-source 1O2 generator that is unlike current PDT methods,
which employ the systemic administration of dyes. The optical
fiber was developed for the site-specific delivery of photosensi-
tizer molecules. Three areas were discussed: (1) A porous fiber
optic cap with photodetachable pheophorbide molecules was
synthesized, and no leaching of the sensitizer was observed in
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Scheme 5
Figure 9. A schematic cross-section picture of the hollow-core fiber
optic, in which ∼350 excitation fibers surround the Teflon inner flow
tube coaxially.
Eclipse TE 200 inverted epifluorescence microscope. Optical images of
the glass samples were collected on an Olympus SZX10 stereo micro-
scope. Some samples were irradiated with a Rayonet photoreactor fitted
with Sylvania visible light bulbs through a 0.05 M NaNO2 filter solution.
Optical Fiber. The apparatus consisted of a 250-W quartz-halogen
illuminator (Fiberoptic Systems, Inc., Simi Valley, CA), a custom-made
fiber-optic cable, a compressed oxygen gas tank, and PVG tip 1. A 3 ft,
0.55 numerical aperture borosilicate fiber optic was used, which had an
external diameter of 1.4 mm including the jacket black polyvinyl
chloride. The fiber optic contained a 1.1 mm diameter Teflon gas flow
tube running from the distal end to the T-valve, which was surrounded
by ∼350 excitation fibers (Figure 9). An integral dichroic reflector was
used to focus the light of the illuminator into the proximal end of the
fiber, and 28 mW was delivered out of the end of the fiber (beam
area =0.126 cm2, energy density of output =444 mJ/cm2). Some
experiments employed the use of a cutoff filter (<400 nm). The T-value
was connected to a 200 PSI compressed oxygen tank with the gas
regulator set at 2 or 10 PSI (flow rate through the cap was 0.2ꢀ0.3 ppm/
min). PVG caps were shaped into cylindrical pieces with a Buehler
IsoMet Low Speed Saw (Model 11-1280-160), a Buehler ultrasonic disk
cutter (Model 170), and a Buehler variable speed grinder-polisher. A
hole (3.0 mm diameter ꢁ 2.0 mm length) was drilled into the PVG
cylinders with a dremel drill (Model 200) to accommodate the fiber
optic, which was glued in place with ethyl cyanoacrylate. Regarding the
propagation of light, most was lost out of the end of the tip rather than
scattered evenly within the tip. The PVG cap received the excitation light
and oxygen gas, but did not heat up; it remained at room temperature
throughout the course of the experiments.
Pheophorbide-Modified Glass (1). Covalent bonding of the
sensitizer to the PVG was achieved by adding pheophorbide monoester
11 (10 mg, 0.0126 mmol) to 3-iodopropyltrimethoxysilane (0.250
mmol) and NaH (0.302 mg, 0.0126 mmol) in 5 mL of dry THF, and
refluxing the mixture at 70 °C for 24 h. THF was evaporated under N2
leaving the pyropheophorbide 12ꢀ3-iodopropyltrimethoxysilane resi-
due, which was then added to 100 mL of dry toluene and twelve 0.2-g
PVG caps (predried at 500 °C in a Fisher Scientific Isotemp muffle
furnace for 24 h) and refluxed at 110 °C for 24 h. The colorless PVG tips
were converted to a deep green color when silane 12 was anchored to the
glass. Control experiments were carried out in order to establish that the
sensitizer molecules derived from 12 were anchored and not adsorbed to
the silanol groups of PVG. Any silanes that were not covalently attached
to the PVG surface were washed away with toluene, THF, chloroform,
ether, and hexane, followed by Soxhlet extraction with methanol for 24 h.
In the absence of 3-iodopropyltrimethoxysilane and NaH, a weak
noncovalent interaction existed between 11 molecules and PVG that
were readily carried off the surface by solvent washing and Soxhlet
extraction. Regarding the dark stability of 1, no leaching of the photo-
sensitizer was observed under the following conditions: (a) 1.5 mL of
0.01 M HCl solution (pH 2) was added to 156 mg of 1 at room
temperature (rt) for 4 h; (b) 1.5 mL of 0.01 M dilute NaOH solution
self-quenching. (3) Pigmentation of semisolid petrolatum was
observed where 100% (60 nmol) sensitizer detached from the
probe tip. However, photorelease was much less efficient in
toluene-d8 and D2O, because 3 remained adsorbed on the probe
tip even though the covalent alkene bond bridging the sensitizer
and glass had been broken.
The results reported here provide knowledge of the factors
influencing sensitizer photorelease for the development of the
new area of fiber optic-guided sensitizer delivery. The potential of
this fiber optic for cancer cell killing in discrete locations remains
to be determined.
’ EXPERIMENTAL SECTION
Materials and Instrumentation. Corning 7930 porous Vycor
glass was purchased from Advanced Glass and Ceramics, Holden, MA.
Pyropheophorbide-a was purchased from Frontier Scientific and was
used as received. Spectrophotometric grade toluene-d8, deuterium
oxide-d2, chloroform-d1 were purchased from Sigma Aldrich or Cam-
bridge Isotope Laboratories and were used as received. Acetonitrile-d3
was purchased from Isotec, Inc. Deionized water was purified using a
U.S. Filter Corporation deionization system. Reagents and solvents such
as NaOH, 4-bromophenol, 1,2-dibromoethane, CH2Cl2, CCl4, benzoyl
peroxide, acetone, NaI, sodium thiosulfate, ethanol, n-BuLi, DMF,
NH4Cl, anhydrous Na2SO4, MeOH, NBS, NaBH4, DMAP, EDC,
NaH, 3-iodopropyltrimethoxysilane, tetramethoxy benzene, toluene,
and THF were purchased from Sigma Aldrich and used without further
purification. Column chromatography was carried out on silica gel
40ꢀ60 Å particles. TLC was conducted on silica gel 60F 254 TLC-
plates. The radiant power of the visible light exiting the fiber was
measured with a Nova energy meter from Ophir Optronics, Logan, UT.
Dissolved oxygen was measured with a Hach sens-ION6 dissolved
oxygen meter. Proton NMR spectra were acquired at 400 MHz and
13C NMR spectra were acquired at 100.6 MHz on a Bruker DPX400
MHz instrument. HRMS data were obtained on an Agilent 6220-TOF
coupled with 1200 series LC. GC/MS data were acquired on an Agilent
6890N coupled with 5973 MSD. HPLC data were obtained on a Perkin-
Elmer 200 series instrument equipped with a bondclone 10 C18 column
at 254 nm and FTIR spectra were collected on a Perkin-Elmer Spotlight
Imaging System. The melting points were obtained on a MEL-TEMP
apparatus. UVꢀvisible spectra were collected on a Hitachi UVꢀvis
U-2001 instrument. Fluorescence spectra were collected on a Nikon
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Journal of the American Chemical Society
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(pH 12) was added to 130 mg of 1 at rt for 4 h. In both cases [(a) and
(b)], the aqueous solution or chloroform extract did not show any trace
of organics in solution by absorption spectroscopy (detection limit of
pheophorbide-a = 10ꢀ8 M)). The heterogeneous sensitizer 1 was
recovered by drying under vacuum at rt for 12 h. UV (air) λ: 507,
538, 610, and 666 nm; FT-IR: 2851 and 2954 cmꢀ1. In 1, the ratio of
sensitizer to iodosilane to silanol groups was ∼1:3:300.
(d, J= 16.0 Hz, 1H), 4.90 (d, J= 12.0 Hz, 1H), 4.61 (s, 2H), 4.46 (d, J=7.08
Hz, 1H), 4.28 (d, J = 8.3 Hz, 1H), 3.70 (m, 4H), 3.40 (s, 3H), 3.24 (s, 3H),
2.61 (m, 2H), 2.31 (m, 2H), 1.78 (d, J = 7.2 Hz, 3H), 1.70 (t, J = 7.6 Hz,
3H), ꢀ1.68 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 11.2, 12.0, 17.4, 19.5,
23.1, 29.7, 29.8, 31.1, 48.0, 49.9, 51.6, 64.8, 65.9, 93.0, 97.2, 104.1, 106.1,
116.1, 116.3, 122.5, 128.1, 128.3, 128.5, 128.6, 129.2, 130.0, 130.1, 130.5,
130.6, 131.5, 135.4, 135.9, 136.1, 136.2, 137.9, 141.5, 145.0, 149.0, 150.8,
155.2, 156.9, 157.3, 160.2, 171.3, 172.8, 196.1. HRMS [þESI] calcd for
C49H48 N4O6 (Mþ) 788.3574, found: 788.3562.
4-Hydroxybenzyl-pyropheophorbide Ester Sensitizer
(13). Pheophorbide ester 3 (2 ꢁ 10ꢀ5 M) was placed in methanolꢀ
water mixtures (9:1) where the pH values ranged from 2 to 8. The pH of
the solution was adjusted by adding 0.01 M NH4OH and 0.01 M
HCOOH. Compounds 13 and 14 were monitored by LCMS: Solvent A
(water containing 0.1% formic acid and 5 mM ammonium formate) and
Solvent B (MeOH containing 0.1% formic acid and 5 mM ammonium
formate) were used for isocratic elution. B (90%) was run through a
30 mm C-18 column for 6 min. HRMS [þESI] calcd for C40H40N4O4
(Mþ) 640.3050, found. 640.3048.
Oxygen Flow Measurements. The rate of oxygen flow through
the porous Vycor cap was measured using a Hach sensION6 dissolved
oxygen meter. The oxygen meter and the fiber optic tip were placed
simultaneously into 30 mL of distilled H2O. Oxygen flowed through the
fiber optic cable at 2 and 10 PSI. Measurements were taken from the
oxygen meter at 1-min intervals for a total of 30 min.
Sensitizer Coverage Measurements. Two methods were con-
ducted to determine the amount of sensitizer bonded to the PVG tip. (i)
The sensitizer was liberated from the PVG surface by dipping 1 into a
30% (v/w) hydrofluoric acid solution for 12 h using a previously
established method.26 The free sensitizer in solution was extracted with
chloroform and its concentration determined by UVꢀvisible spectros-
copy. (ii) A calculation was performed to determine the amount of 11 or
12 remaining in toluene solution after PVG was removed in 12 h with a
correction for any adsorbed sensitizer by Soxhlet extraction. Even
though millimole amounts of silane could be loaded due to the available
silanol groups (1.66 mmol SiOH/g PVG), the range of sensitizer silane
12 loaded was 0.06ꢀ1.1 μmol.
Photocleaved 4-(Formyloxy)benzyl-pyropheophorbide
Ester Sensitizer (3). One gram of pheophorbide-modified glass 1
(3 ꢁ 10ꢀ7 mol/g) was taken into 1 mL of toluene-d8 solution. The
solution was purged with oxygen for 10 min. The heterogeneous
solution was irradiated with the Rayonet reactor for 2 h. The photo-
cleaved compound 3 was analyzed by following LCꢀMS condition:
Solvent A (water containing 0.1% formic acid and 5 mM ammonium
formate) and Solvent B (MeOH containing 0.1% formic acid and 5 mM
ammonium formate) were used for isocratic elution. B (90%) was
delivered through the 30 mm C-18 column for 6 min wherein compound
3 was found to elute at 2.3 min. For the heterogeneous experiments,
Soxhlet extraction was conducted to quantitate the pheophorbide
formate ester (3) that detached from the silica matrix HRMS [þESI]
calcd C41H40N4O5 (Mþ) 688.2999, found 688.2997.
(Z)-4,40-(Ethene-1,2-diylbis(oxy))dibenzaldehyde (9). Yield
0.30 g (60%). To 0.5 g of arylbromide 8 in 5 mL of THF, 1.6 mL of
(0.003 mol) n-BuLi was added and the solution was stirred at ꢀ78 °C for
0.5 h. DMF (0.0058 mol, 0.5 mL) was added to the reaction mixture and
stirred for 20 min at ꢀ78 °C. The solution was warmed to room
temperature and was stirred for an additional 3.5 h. The reaction mixture
was quenched with 20 mL of cold saturated aqueous NH4Cl and then
25 mL of CH2Cl2 was added to the solution. The organic layer was
washed with saturated sodium chloride solution and dried with Na2SO4.
The crude liquid was purified by column chromatography (3:1 hexane
and ethyl acetate) to yield 9 (mp 108ꢀ110 °C). Rf = 0.15; 1H NMR (400
MHz, CDCl3) δ 9.93 (s, 2H), 7.88 (d, J = 8.8 Hz, 4H), 7.20 (d, J = 8.8
Hz, 4H), 6.34 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 116.2, 128.4,
131.8, 131.9, 161.5, 190.5. HRMS [þESI] calcd for C16H12O4 (Mþ)
268.0736, found: 268.0732.
(Z)-(4,40-(Ethene-1,2-diylbis(oxy))bis(1,4-phenylene))di-
methanol (10). Yield 97 mg (100%). To 100 mg (0.37 mmol) of 9 in
2.5 mL of MeOH, 0.140 g (1.86 mmol) of NaBH4 was added in small
proportions and the reaction mixture was stirred for 14 h. Methanol
solvent was then evaporated and the reaction was quenched with
saturated aqueous NH4Cl solution. Ethyl acetate was then added to
the solution mixture and the organic layer was separated and dried over
Na2SO4. Evaporation of solvent gave solid 10 (mp: 150ꢀ153 °C). The
acid stability of the alkene linkage of bis-alcohol 10 was examined: 0.02 M
10 was added to 0.02 M HCl (pH 2) with 1.1 ꢁ 10ꢀ4 M 1-pentanol as the
internal standard with stirring in 2 mL of CDCl3. The solution was stirred
and monitored by 1H NMR after 0, 60, and 120 min with no degradation
or loss of the alkene peaks. 1H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 8.4
Hz, 4H), 7.09 (d, J = 8.4 Hz, 4H), 6.15 (s, 2H), 4.66 (d, J = 3.9 Hz, 4H).
13C NMR (100 MHz, CDCl3) δ 64.3, 116.1, 128.3, 128.4, 135.5, 156.8.
HRMS [þESI] calcd for C16H16O4 (Mþ) 272.1049, found: 272.1047.
(Z)-4-[2-(4-Hydroxymethyl-phenoxy)-vinyloxy]benzyl-pyro-
pheophorbide (11). Yield 6 mg (60%). Ten milligrams (0.018 mmol)
of commercially available pyropheophorbide-a, 5.0 mg (0.018 mmol) of bis-
alcohol 10, 2.28 mg (0.018 mmol) of DMAP, and 5.37 mg (6.9 mmol) of
EDC were added into 3 mL of DCM. After stirring overnight at room
temperature, DCM was evaporated from the reaction mixture and the crude
product was purified by column chromatography (1% methanol in CHCl3)
yielding a black solid. Rf = 0.48. 1H NMR (400 MHz, CDCl3) δ9.51 (s, 1H),
9.40 (s, 1H), 8.54 (s, 1H), 8.01 (dd, J = 17.8, 11.5 Hz, 1H), 7.29 (d, J = 8.5
Hz, 2H), 7.14 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.5
Hz, 2H), 6.28 (d, J = 17.8 Hz, 1H), 6.17 (d, J = 11.5 Hz, 1H), 6.08 (dd, J =
17.0, 3.4 Hz, 2H), 5.24 (d, J = 19.8 Hz, 1H), 5.06 (d, J = 19.8 Hz, 1H), 4.98
Photocleavage Procedure. The 0.2 g fiber optic cap 1 (60 nmol
12) was placed into 0.5 mL of toluene-d8 or 1.0 mL of D2O, 0.4 mL of
petrolatum at 65 °C. The amount of photocleaved 3 was determined by
absorption spectroscopy for the toluene-d8 and D2O samples, and by
fluorescence spectroscopy for the petrolatum samples. The fiber tip was
secured and then the cap was irradiated via the fiber with white light for
4 h. A TXRed optical filter was used and images were created with
Metafluor imaging software. Concentrations of dye were calculated by
measuring the fluorescence intensity and comparing values to those
found in the photorelease of dye from neat glass caps. The diffusion of
pyropheophorbide 3 away from the probe tip into petrolatum was
viewed using the epifluorescence microscope and was measured with the
Adobe Photoshop CS5 ruler tool. The experiment was performed for a
total of 2 h and images were taken every 30 min at 4ꢁ and 10ꢁ
magnification.
SensitizerꢀSensitizer Surface Distance Calculation. PVG is
a transparent material with ∼4 nm diameter pores and a ∼250 m2/g
surface area, interconnected pores, and the surface has “stalagmite-like”
features running ∼3 nm in length and height.16 It is difficult to accurately
calculate the spatial distance between sensitizer molecules in the porous
glass matrix; however, a likely distance was estimated. With an average
distance between silanol groups of 10 Å, the calculation (4 ꢁ 250 m2/g)/
100 Å2 = 1 ꢁ 1021 SiꢀOH groups is divided by Avogadro’s number =
1.66 ꢁ 10ꢀ3 mol SiꢀOH/g PVG. Equations 1ꢀ4 show how a simple
surface geometry wasassumed. Equation 1 givesthevolume of1 within the
0.08 mmsensitizerintrusiondepth of the glassofradius (r) and height (h).
The weight of the sensitizer-intruded portion of glass was calculated
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Journal of the American Chemical Society
ARTICLE
multiplying its volume by its density (1.38 g/mL). The area of the
sensitizer-intruded glass is given by eq 2. The area occupied by 4 sensitizer
molecules at a given loading amount (0.06ꢀ1.1 μmol 12) is given by eq 3.
Dye molecules were assumed to be spread out with an orthogonal
orientation in relation to the surface. The sensitizerꢀsensitizer distance
on the PVG surface is given by eq 4.
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vol: ðmm3Þ ¼ ½ðπ ꢁ r12 ꢁ h1Þ ꢀ ðπ ꢁ r22 ꢁ h2Þꢂ
glass area ðÅ2Þ
ð1Þ
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ðmols Si-OHÞ ꢁ ð6:023 ꢁ 1023 Si-OH groups=molsÞ ꢁ ð100 Å2Þ
¼
4Si-OH groups
ð2Þ
ꢀ
ꢁ
ðglass area ꢁ 4Þ
area between 4 sensitizer sites ðÅ2Þ ¼
ð3Þ
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sensitizer molecules
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
sensitizer ꢀ sensitizer dist: ðÅÞ ¼ ðarea of 4 sensitizer molecules Å2Þ
ð4Þ
’ ASSOCIATED CONTENT
S
Supporting Information. Details of the syntheses of
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’ AUTHOR INFORMATION
Corresponding Author
agreer@brooklyn.cuny.edu
ꢁ
Nature 2007, 447, 273–274. (j) Baruah, A.; Simkovꢀa, K.; Hincha, D. K.;
Apel, K.; Laloi, C. Plant J. 2009, 60, 22–32.
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We acknowledge support from the National Institute of
General Medical Sciences (NIH SC1GM093830). A.G. is thank-
ful to Zhong Wang (Hunter College Bio-Imaging Facility), Terry
Dowd (Brooklyn College Chemistry Department), and Mim
Nakarmi (Brooklyn College Physics Department) for use of
requisite equipment.
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