Two-photon absorbing block copolymer as a nanocarrier for porphyrin: Energy transfer and singlet oxygen generation in micellar aqueous solution

Article abstract of DOI:10.1021/ja071057p

A novel amphiphilic water-soluble 2PA-chromophore-containing block copolymer was designed and synthesized. Micelles (nanostructures) were generated in the aqueous solution using the block copolymer and characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM). We demonstrated that hydrophobic porphyrin could be incorporated into aqueous solution with the assistance of micelles. The results from DLS showed that the average diameters are in the range of 59-77 nm. Efficient energy transfer (as high as 96%) from the 2PA chromophore to porphyrin was observed in micellar aqueous solution even though the donor and acceptor were not covalently bonded. Furthermore, the energy transfer contributes to the efficient generation of the singlet oxygen (by a factor of 2.6 at 800 nm) from the porphyrin guest molecules. Copyright

Full text of DOI:10.1021/ja071057p

Published on Web 05/17/2007
Two-Photon Absorbing Block Copolymer as a Nanocarrier for Porphyrin:
Energy Transfer and Singlet Oxygen Generation in Micellar Aqueous Solution
Ching-Yi Chen, Yanqing Tian, Yen-Ju Cheng, A. Cody Young, Jae-Won Ka, and Alex K.-Y. Jen*
Department of Materials Science and Engineering, UniVersity of Washington, Seattle, Washington 98195-2120
Received February 14, 2007; E-mail: ajen@u.washington.edu
Porphyrin-based derivatives are widely used as photosensitizers
for singlet oxygen (1O2) generation, which is a key step for
photodynamic therapy.1 However, most porphyrins absorb light in
the visible region and have relatively low two-photon absorption
(2PA) cross-sections (δ: 1-10 GM; 1 GM ) 10-50 cm4
s
photon-1).2 These characteristics limit their effectiveness when
excited with a near-infrared (NIR) light source (ranging from 700
to 1000 nm), which would allow deeper penetration and less damage
to biological tissues.3 Direct chemical modification of porphyrins
by preparing porphyrin dimers and π-conjugated porphyrins4 is a
commonly used approach to solve these problems. Alternatively,
the utilization of fluorescence resonance energy transfer (FRET)5
in light harvesting dendrimers6,7 and silica nanoparticles8 is also
quite effective. In these dendrimers, the peripheral donors possess
a relatively large two-photon cross-section that can efficiently
absorb NIR light and transfer the energy to the porphyrin core. As
a result, these porphyrin-containing dendrimers could be used to
Figure 1. The chemical structures of P1, P2, and 1.
Table 1. Typical Physical Properties of P1 and P2
ET (%)
Average sizes of the micelles
systems
ratioa
1PF b
2PF c
AMd
DLSf
AFMg
P1
0
0
e
2.7
6.5
4.4
59 (0.14)
66 (0.14)
67 (0.13)
70 (0.14)
73 (0.14)
77 (0.18)
53 ( 11
50 ( 12
48 ( 10
52 ( 11
52 ( 15
54 ( 14
1
efficiently generate O2.6
1/P1
1/P1
1/P1
1/P1
1/P2
0.1
0.2
0.6
1.0
1.0
47
65
86
91
50
71
89
96
Herein, we report the use of a new amphiphilic 2PA-chro-
mophore-containing block copolymer to enhance the efficiency of
1O2 generation. This is realized by using the block copolymer (P1,
Figure 1) as a nanocarrier to encapsulate a hydrophobic porphyrin
photosensitizer (1, Figure 1) inside its micelles. In aqueous solution,
P1 forms micelles with the hydrophilic poly(ethylene glycol) (PEG)
as the corona to ensure good water solubility. The 2PA-chro-
mophore-containing hydrophobic core provides a good environment
to accommodate the hydrophobic 1. Due to the size confinement
in these micelles, efficient FRET from the 2PA chromophore to
porphyrin occurs, which enhances the effective 2PA cross-sections
of the porphyrin.
This approach combines several advantages: (1) numerous
photosensitizers can be easily incorporated into an aqueous system
without encountering tedious chemical modifications; (2) biocom-
patible 2PA block copolymers can be used as nanocarriers for
bioapplications; and (3) the FRET through noncovalently bonded
donors and acceptors within micelles enhances the efficiency of
1O2 generation under NIR light irradiation.
The structures of two block copolymers synthesized (P1 and P2)
are shown in Figure 1. In order to reduce chromophore aggregation
within the micelles, the 2PA monomer (M) was copolymerized with
styrene to produce P1.9 The aryl amino-containing donors were
connected with a fluorene bridge through triple bonds to enhance
its photostability.10 To differentiate the contribution of FRET to
1O2 generation, P2 without the 2PA chromophore was also
synthesized for comparison.
Micelles were prepared in aqueous solution using the typical
dialysis method and were characterized by dynamic light scattering
(DLS) and atomic force microscopy (AFM). The average diameters
of the micelles are listed in Table 1.
a Molar ratio of 1 to P1 or P2 determined by UV-vis spectra. b Energy
transfer efficiency irradiated at 380 nm. c Energy transfer efficiency excited
at 800 nm. d Amplification using P2 as counterpart excited at 800 nm.
Integrated emission intensity of 1 was collected from 625 to 750 nm. e The
signal is too weak to detect. f Determined by dynamic light scattering in
solution at 25 °C. The data in the parentheses are the polydispersities.
g Observed by AFM using dried sample on mica surface.
Information for details), indicating that efficient FRET can occur
from the 2PA chromophore to 1. Sensitizer 1 is insoluble and does
not fluoresce in an aqueous solution. However, once it was
encapsulated within a micelle, the aqueous solution began to
fluoresce. For this study, the concentration of P1 was kept constant
(0.075 mg/mL, which corresponds to a 2PA chromophore concen-
tration of 7.5 µM). When the concentration of 1 in P1 (simplified
as 1/P1) was increased, the fluorescence intensity of the 2PA
chromophore decreases (Figure 2A). The energy transfer efficiency
can be calculated to be as high as 91%.11 This indicates that effective
FRET indeed occurs within these micelles.12 It also proves that
the nanoconfinement provided by the 2PA block copolymer helps
efficiently generate the porphyrin excited state through energy
transfer.
P1 in aqueous solution exhibits 2PA cross-sections of 288 GM
at 750 nm and 120 GM at 800 nm when excited with a femtosecond
mode lock Ti-sapphire laser. Although these values are moderate
compared to other values reported in the literature,13 they are still
much higher than those from the porphyrins alone. The energy
transfer efficiencies of 1/P1 excited at 800 nm are similar to those
excited at 380 nm (Table 1). Furthermore, the emissions of 1 in
1/P1 through FRET are 2.7-6.514 times higher than the emissions
The fluorescence spectrum of the 2PA chromophore overlaps
with both the Soret and Q-band absorptions of 1 (see Supporting
9
7220
J. AM. CHEM. SOC. 2007, 129, 7220-7221
10.1021/ja071057p CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
under the Bio-inspired Concept, the Microscale Life Sciences Center
(an NIH Center of Excellence), and the Boeing-Johnson Foundation
is acknowledged.
Supporting Information Available: Syntheses and experimental
details, UV-vis spectra, fluorescence spectra, DLS and AFM images
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
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Figure 2. (A) The fluorescence spectra of 1 in P1 with various loading
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emission of 1 in P1 (f) and in P2 (g) with the ratio of 1/1 irradiated at 800
nm. (C) The typical photochemical process of 1/P1/ADPA under the
excitation of 800 nm in D2O. The absorbance of 1 at 420 and 515 nm has
no change, indicating the stability of 1 under the experimental condition.
(D) The first-order plots for the photooxidation of ADPA in 1/P1/ADPA
(curve h), 1/P2/ADPA (curve i), and P1/ADPA (curve j) against irradiation
time. A0 is the absorbance at 378 nm before irradiation. A is the absorbance
after irradiation. The ratio of 1 to P1 or P2 in mole is 0.6/1
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of 1 in the 1/P2 systems because no 2PA-chromophore-enhanced
FRET was involved in 1/P2 (Table 1 and Figure 2B). This confirms
that efficient FRET only occurs with the aid of 2PA chromophores.
The ability of the system to generate singlet oxygen from
porphyrin was evaluated using the disodium salt of 9,10-an-
thracenedipropionic acid (ADPA) as a water-soluble 1O2 sensor.15
The generated 1O2 can react with ADPA which results in bleaching
ADPA to its corresponding endoperoxide. The progress of the
reaction could be monitored by observing the decrease of the ADPA
absorption intensity at 378 nm when pumped with an 800 nm light.
For comparison, three different oxygen-saturated aqueous solutions16
(1/P1/ADPA, 1/P2/ADPA, P1/ADPA) with the same concentration
of ADPA and/or 1 were studied. Without 1, almost no ADPA
absorption intensity change was observed throughout the time (curve
j), indicating that bleaching of ADPA by the irradiating light itself
was negligible. A noticeable time-dependent decrease in the ADPA
absorbance in mixtures containing 1 was observed (Figure 2C).
This observation shows both the generation of 1O2 and its
subsequent diffusion out of micelles. The values of the observed
rate constants of 1/P2/ADPA and 1/P1/ADPA are (1.41 ( 0.2) ×
10-3 and (3.69 ( 0.3) × 10-3 min-1, respectively. Thus, the
bleaching rate of the system with the 2PA chromophore is 2.6-
fold faster than that without the 2PA chromophore. This confirms
that the FRET through the 2PA chromophore contributes signifi-
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observed, resulting in the low quantum yield in micellar water (0.39) as
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was observed in their THF mixtures without micellar structures, possibly
due to charge or electron transfer. However, these quenching effects were
much smaller than those in the micelles. Thus, we believe that the size
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1
cantly to the O2 generation.
In conclusion, a novel water-soluble amphiphilic 2PA-chro-
mophore-containing block copolymer was prepared. Hydrophobic
porphyrin was incorporated into an aqueous solution with the
assistance of micelles. Efficient energy transfer (as high as 96%)
from the 2PA chromophore to porphyrin was observed in micellar
aqueous solutions under the 2PA conditions. Furthermore, the
efficient energy transfer contributes to effective singlet oxygen
generation (by a factor of 2.6 at 800 nm) from the porphyrin guest
molecules. We have developed a new strategy to use covalently
bonded donors to trap acceptors within an ideal nanoenvironment
for improving the efficiency of singlet oxygen generation under
NIR light irradiation in aqueous solution. This advancement may
generate further interest in developing efficient two-photon block
copolymers for bioapplications.
(14) The amplification of 1/P1 determined by using 1/P2 as counterpart is
affected by two factors: energy transfer efficiency and concentration of
1 within the micelles. The large amplification is observed in the molar
ratio of 0.6/1 due to the fact that it has similar energy transfer efficiency
but less aggregation of 1 within the micelles compared to that of a
molecular ratio of 1/1.
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(16) These experiments were performed by adding 25 µL of ADPA (2 mg/
mL) into 2.5 mL of the micellar solutions of P1, 1/P1, and 1/P2 with the
same concentration of block copolymer and/or 1 at room temperature.
Here, D2O was used to replace H2O due to the concern of the much longer
1
Acknowledgment. Financial support from the National Science
Foundation (NSF-STC Program under Agreement Number DMR-
0120967), the Air Force office of Scientific Research (AFOSR)
lifetime of the O2 in D2O. The solution was bubbled with oxygen for 5
min to get an oxygen-saturated aqueous solution.
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