Controlled generation of singlet oxygen by porphyrin-appended gold nanoparticles

Article abstract of DOI:10.1246/bcsj.20160254

Porphyrin-appended gold nanoparticles with different chain lengths were synthesized to examine the control over photosensitization. The efficiencies evaluated by singletoxygen generation were adjusted by the average number of porphyrins on one gold nanoparticle and the particle size regardless of the linker chain length between porphyrin site and gold core.

Full text of DOI:10.1246/bcsj.20160254

Advance Publication Cover Page
Controlled Generation of Singlet Oxygen by Porphyrin-Appended Gold
Nanoparticles
Akira Shinohara and Hideyuki Shinmori*
Advance Publication on the web August 26, 2016
doi:10.1246/bcsj.20160254
©
2016 The Chemical Society of Japan

Short Articles
moieties. Thus, intraparticle ligand-ligand interactions and
ligand-nanoparticle interactions could not be ignorable. At
least to our best knowledge, there is no report about the
quantitative evaluation of photosensitization efficiency of
porphyrin-appended gold nanoparticles. In this article, we
provide basic information about the concentration effects of
porphyrin moieties on the photosensitization efficiency of
porphyrin-appended gold nanoparticles with different chain
lengths.
Controlled generation of singlet oxygen
by
porphyrin-appended
gold
nanoparticles
Akira Shinohara and Hideyuki Shinmori*
We employed two different size (diameter)
Interdisciplinary Graduate School of Medicine and Engineering,
University of Yamanashi, 4-4-37 Takeda, Kofu 400-8510
1
(
-dodecanethiolate-protected
gold
nanoparticles
C12SAuNP) ; as-synthesized 2.5±0.9 nm nanoparticles
3
prepared using the Brust-Schiffrin methodology which show
only weak LSPR on their UV/vis absorption spectrum, and
LSPR active (~520 nm) 5.6±2.2 nm nanoparticles obtained by
heat-induced size evolution of nanoparticles according to a
Received XX XX, 20XX; E-mail: shinmori@yamanashi.ac.jp
5
Porphyrin-appended gold nanoparticles with different
chain lengths were synthesized to examine the controlling
over photosensitization. The efficiencies were evaluated by
singlet oxygen generation under visible light irradiation,
and were adjusted by the average number of porphyrins on
one gold nanoparticle and the particle size regardless of the
linker chain length between porphyrin site and gold core.
literature method reported by Miyake et al.. Each of the mean
diameters calculated by counting the sizes of about 200
particles and particle size distribution were determined from
TEM images (see the Supporting Information, Figure.S1). We
also synthesized three porphyrin-appended alkanethiols
PC2SH, PC5SH, and PC10SH, which have a different
methylene linker chain length (n = 2, 5, and 10) attached by
amide bond, and then porphyrin-appended alkanethiols
reacted with above two different size C12SAuNP (i. e. 2.5 ±
Photosensitization is a photochemical reaction that
forms a key process in photodynamic therapy (PDT), in which
singlet oxygen is generated by the energy transfer from the
excited states of photosensitizer to the ground state of
0
.9 nm and 5.6
±
2.2 nm) to obtain the six
porphyrin-appended gold nanoparticle species (2.5 nm
PC2SAuNP, 2.5 nm PC5SAuNP, 2.5 nm PC10SAuNP, 5.6
1
molecular oxygen in tissue. Singlet oxygen, which is one of
the reactive oxygen species (ROS), causes irreversible
damages of various cellular organelles and biomolecules,
including mitochondria, protein, lipid, nucleus, etc. Recently,
the controlling of photosensitization efficiency of
photosensitizer has attracted great attentions because of
undesired post-treatment phototoxicity by remaining drugs in
PDT. The reducing phototoxicity following therapy is of
importance to become a standard treatment for tumors.
+
meso-Tetraphenylporphyrin-appended
alkanethiols with three different chain lengths
PCnSH
1-Dodecanethiolate-protected AuNPs with
two different core diameters
C12SAuNP
(n = 2, 5, 10)
(2.5 ± 0.9 nm and 5.6 ± 2.2 nm)
Thiolate/thiol ligand exchange reaction
Toluene, r. t., 5 – 90 min
Porphyrins and other tetrapyrrolic macrocycles are
commonly used in PDT for cancer treatments. Although
porphyrin is well-known to be a strong singlet oxygen
2
generator, it has been difficult to tune their photosensitization.
A promising approach for overcoming the drawbacks is the
energy transfer from excited porphyrins to another acceptor.
On the other hand, metal nanoparticles have captured the
interest of a wide range of scientists, since their physical
properties are intrinsically different from those of the bulk
substances depending upon their size and shape. In particular,
alkanethiolate-protected gold nanoparticles, developed in the
2
.5 nm PCnSAuNP
5.6 nm PCnSAuNP
Scheme 1. Synthesis of six
porphyrin-appended gold nanoparticles.
nm PC2SAuNP, 5.6 nm PC5SAuNP, and 5.6 nm
PC10SAuNP) as shown in Scheme 1.
Thiolate/thiol ligand exchange reaction was performed
at ambient temperature in homogeneous toluene solution. A
solution of 1-dodecanethiolate-protected gold nanoparticles
3
past quarter of century by Brust et al., are considered as ideal
candidates for the components of novel photofunctional
4
materials because of their great flexibilities, such as
extraordinary stability, ease of functionalization by
thiolate/thiol ligand exchange reaction, their large specific
surface area, and their own interesting photoproperties caused
by localized surface plasmon resonance effect (LSPR).
Additionally, they can be dispersed in various solvents even in
water by pre- or post-synthesis for surface modifications to
give them desired solubility. Substitution degree on particle
surface (i. e. the number of functional ligands per particle)
seems significant if we need quantitative discussions about
photochemical properties of introduced photofunctional
(
10 mg/mL) was added PCnSH (5 mg/mL). The progress of
ligand exchange from the 1-dodecanethiolate to the
porphyrin-appended alkanethiolate was monitored by UV/vis
spectroscopy. The reaction was quenched at certain times (5,
0, 15, 30, and 60 min for 2.5±0.9 nm, and 10, 20, 30, 60, and
0 min for 5.6±2.2 nm particle core) by injection of aliquot
0.2 mL) into excess acetone (40 mL) to precipitate
porphyrin-appended gold nanoparticles. After centrifugation
10,000 × g), the purple supernatant containing the
1
9
(
(

1
0
5
0
40
A
(2.5 nm PC2SAuNP)
B
(5.6nm PC2SAuNP)
3
0
90 min
6
0 min
20
10 min
5
min
1
0
0
3
00
400
500
Wavelength [nm]
600
700
3
00
400
500
Wavelength [nm]
600
700
Figure 1. Time course of UV/vis extinction evolution of porpyrin-appended gold nanoparticles in toluene (A) 2.5 nm
PC2AuNP (B) 5.6 nm PC2AuNP after purification (i. e. precipitation, SEC purification)..
unbounded alkanethiol-appended porphyrins was discarded,
and the black precipitate was applied to size exclusion
chromatography in order to accomplish the complete removal
of a late-eluted purple fraction containing the unbounded
porphyrin ligands. A fast-eluted brown fraction was collected
and was subjected to further characterization. The
representative time courses of UV/vis extinction evolution of
porpyrin-appended gold nanoparticles 2.5 nm PC2SAuNP
and 5.6 nm PC2SAuNP after purification are shown in Figure.
4
0
0
0
2
2
2
2
5
5
.5 nm PC2SAuNP
.5 nm PC5SAuNP
.5 nm PC10SAuNP
.6 nm PC2SAuNP
.6 nm PC5SAuNP
1
. As a result, the porphyrin-appended gold nanoparticles that
have obvious absorption bands arisen from porphyrin moieties
were obtained in a few minutes of the ligand exchange
reaction. Both the porphyrin-appended gold nanoparticles
showed structureless absorption bands from gold nanoparticle
core containing distinct bands from porphyrin moieties known
as typical Soret band at 421 nm and Q bands at 519, 554, 595,
and 651 nm, undoubtedly supporting the adsorption of
porphyrin moieties on the gold nanoparticle surface. These
normalized spectra reveal that spectral profiles of gold
nanoparticle do not affected by ligand exchange from
5.6 nm PC10SAuNP
0
20
40
60
80
100
Reaction time (min)
Figure 2. Plots of substitution number of
porpyrin-appended gold nanoparticles 2.5 nm
PCnSAuNP and 5.6 nm PCnSAuNP vs. time of
ligand exchange reaction at room temperature;
[
C12SAuNP] = 10 mg/mL, [PCnSH] = 5 mg/mL,
1
-dodecanethiolates to PC2S groups. Actually, the absorption
in toluene.
spectra for PCnSAuNPs were nearly identical to the sum of
absorption spectra of PCnSH and localized surface plasmon
resonance band of each of the gold nanoparticles (see the
Supporting Information, Figure.S2). Consequently, the molar
ratios of porphyrin to particle, that is, “substitution number”
Δ
absorbed by porphyrin moieties. For the determination of Φ ,
,3-diphenylisobenzofuran (DPBF) was used as a chemical
singlet oxygen quencher in toluene under visible light
irradiation (500 W xenon short arc lamp equipped with optical
Δ
filters (Y-50 and C-50S) which transmit 500-700 nm). Φ can
1
(the average number of porphyrins on one particle) could be
estimated by means of nonlinear least-squares curve fitting for
absorption spectra, assuming that the molar extinction
coefficients of the porphyrin moiety were the same as those of
PCnSH. The values were increased along with the increasing
of ligand exchange reaction time according to quasi
second-order manner (Figure 2). In all cases with different
chain lengths, the porhyrin substitution numbers for 5.6 nm
PCnSAuNP were invariably several times larger than those
for 2.5 nm PCnSAuNP. This may result from the radius of
curvature of a particle core. Namely molecule loading density
on surface is higher in large particles due to its lower
be estimated from the decay (decomposition) rate of DPBF
under visible light irradiation according to the following
equation :
7
where ΦΔref is a quantum yield for singlet oxygen generation
of reference compound, namely, meso-tetraphenylporphyrin
8
TPP (ΦΔref = 0.70) ; v and vref are the rate of DPBF decay by
visible light irradiation in the case of porphyrin-appended gold
nanoparticles and reference compound, respectively; n and nref
are the number of photons absorbed by porphyrin moieties on
nanoparticles and reference compound, respectively (nref/n is
relative absorptivity). 90 μM DPBF solution was added into
the suspension of purified porpyrin-appended gold
nanoparticles 2.5 nm PCnSAuNP and 5.6 nm PCnSAuNP,
and then was irradiated with visible light until about 5 %
6
curvature radii.
In photosensitization by type II mechanism, energy
transfer from the excited triplet state of sensitizer to the
oxygen leads to singlet oxygen generation. Singlet oxygen
generation efficiency Φ
Δ
is defined as ratio of the number of
generated singlet oxygen molecules to the number of photons

Short Articles
decay (10-25 min) of absorbance at 416 nm (λmax of DPBF)
monitored by UV/vis spectrometer. As shown in Figure 3, the
In conclusion, we have established synthesis of a series
of porphyrin appended gold nanoparticles with different chain
lengths and substitution numbers by thiolate/thiol ligand
exchange reaction. Singlet oxygen generation efficiency was
influenced by gold nanoparticle core size and substitution
number of porphyrins, although less depended on the alkyl
chain length. The porphyrin-appended gold nanoparticles
developed in this study may be promising as a new agent
capable of controlling photosensitization.
estimated values of Φ
Δ
for six porphyrin-appended gold
nanoparticles (2.5 nm PCnSAuNP and 5.6 nm PCnSAuNP)
decreased with an increase of substitution number, in spite of
low proportion for ligand exchange (< ca. 10 %). However, Φ
Δ
in mixed systems of C12SAuNP (2.5 ± 0.9 nm and 5.6 ± 2.2
nm) and TPP was not changed with the concentration of TPP
in the range 0.05-0.25 μM corresponding to substitution
number in our system (data not shown). This means that
concentration effect of porphyrin units on particle affects
photosensitization efficiency, probably due to existence of
neighboring porphyrin units driven by immobilization onto
We gratefully acknowledge the support of this study by
JSPS KAKENHI Grant Number JP26390002 (H.S.).
particles. It is notable that Φ
Δ
for 5.6 nm PCnSAuNP ever
Supporting Information
showed ~5 fold lager than that of 2.5 nm PCnSAuNP. The
Synthesis of PCnSH, C12SAuNP and PCnSAuNP,
TEM images and UV/vis absorption spectra of C12SAuNP,
experimental details for determination of substitution number
of porphyrin moieties and measurement of singlet oxygen
generation efficiency. This materials available on
http://dx.doi.org/XXX/bcsj.XXX.
phenomenon could be explained by electromagnetic field
9
enhancement by LSPR for gold nanoparticle core which is
well-known as enhancement of fluorescence, Raman
scattering, photoabsorption, etc. The difference of
photosensitization efficiency may be ascribed to the
contribution of localized plasmonic field, which is enhanced
1
0
with the increase in particle size. Also, Φ
Δ
for both 2.5 nm
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Substitution number
Figure 3. Singlet oxygen generation efficiency Φ
Δ
of
porphyrin-appended gold nanoparticles in toluene.

Graphical Abstract
Controlled generation of singlet oxygen by porphyrin-appended gold nanoparticles
A. Shinohara and H. Shinmori
Porphyrin-appended gold nanoparticles with different chain lengths were prepared by the ligand exchange
reaction. Their photosensitization efficiency was estimated as the quantum yield for singlet oxygen generation
under visible light irradiation and the controlled generation ability was investigated.