molecules
ArticleNear-Infrared Light Irradiation of Porphyrin-Modified Gold
Nanoparticles Promotes Cancer-Cell-Specific Cytotoxicity
Hiromi Kurokawa 1,2, *, Atsushi Taninaka 3,4 , Toru Yoshitomi 5 , Hidemi Shigekawa 3 and Hirofumi Matsui 1
1
2
3

4
5

*



Citation: Kurokawa, H.; Taninaka, A.;
Yoshitomi, T.; Shigekawa, H.; Matsui,
H. Near-Infrared Light Irradiation ofPorphyrin-Modified Gold
Nanoparticles Promotes
Cancer-Cell-Specific Cytotoxicity.

Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan; hmatsui@md.tsukuba.ac.jp
MoBiol Technologies Corporation, Tsukuba 305-0031, Japan
Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba 305-8573, Japan;
jun_t@bk.tsukuba.ac.jp (A.T.); hidemi@bk.tsukuba.ac.jp (H.S.)
TAKANO Co., Ltd., Nagano 399-4301, Japan
Research Center for Functional Materials, National Institute for Materials Science, Tsukuba 305-0044, Japan;
yoshitomi.toru@nims.go.jp
Correspondence: hkurokawa.tt@md.tsukuba.ac.jp

Abstract: The use of nanoparticles has been investigated as a new cancer treatment. These can
induce specific cytotoxicity in cancer cells. In particular, Au nanoparticles (AuNPs) have uniquecharacteristics. The maximum absorption spectrum of AuNPs can be adjusted to modify their size or
shape to absorb near-infrared light that can penetrate into tissue without photodamage. Thus, thecombination of AuNPs and near-infrared light can be used to treat cancer in deep-seated organs. To
obtain effective cancer-specific accumulation of AuNPs, we focused on porphyrin and synthesized
a porphyrin-attached Au compound: Au-HpD. In this study, we investigated whether Au-HpD
possesses cancer-specific accumulation and cytotoxicity. Intracellular Au-HpD accumulation was
higher in cancer cells than in normal cells. In order to analyze the cytotoxicity induced by Au-HpD,cancer cells and normal cells were co-cultured in the presence of Au-HpD; then, they were subjected
to 870 nm laser irradiation. We observed that, after laser irradiation, cancer cells showed significant
morphological changes, such as chromatin condensation and nuclear fragmentation indicative of cell
apoptosis. This strong effect was not observed when normal cells were irradiated. Moreover, cancer
cells underwent cell apoptosis with combination therapy.

Molecules 2022, 27, 1238. https://doi.org/10.3390/molecules27041238

Keywords: photodynamic therapy; Au-HpD; nearinfrared; reactive oxygen species

Academic Editors: Carlos J. P.
Monteiro, Catarina I. V. Ramos and
M. Amparo F. Faustino

1. Introduction
Received: 31 December 2021
Accepted: 10 February 2022
Published: 12 February 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

A WHO report shows that the leading cause of mortality worldwide is ischemic heartdisease, followed by cancer. The number of new cancer patients in 2018 was calculated
to be 18 million. Moreover, it is estimated that cancer will become the leading causeof mortality in 2060. Lung, liver, and stomach cancers are the top lethal cancers in thegeneral population [1]. The traditional and latest treatments for cancer include surgery,chemotherapy, and radiotherapy, but all of these treatments cause major side effects innormal tissues. Moreover, some cancers have better prognoses, while other cancers, suchas pancreatic cancer, have worse prognoses. Consequently, the optimal cancer treatment
will have high, cancer-specific toxicity and a low side effect profile for normal tissues [2].
The use of nanoparticles, including Au nanoparticles (AuNPs), has been investigatedas a new cancer treatment that induces specific cytotoxicity in cancer cells [3]. AuNPs haveunique characteristics, such as high biocompatibility, low cytotoxicity for normal tissues,and simple synthesis [4]. Based on these characteristics, many researchers have investigatednew cancer therapies using these particles [5,6]. AuNPs enhance reactive oxygen species(ROS) generation in the following two ways: thermal effect and localized surface plasmon
resonance [3,4]. The maximum absorption spectrum of AuNPs can be adjusted to modify
their size or shape to absorb near-infrared (NIR) light that can penetrate into tissue without

Molecules 2022, 27, 1238. https://doi.org/10.3390/molecules27041238

https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 1238

2 of 10

photodamage. Thus, the combination of AuNPs and NIR light can be used to treat cancer
in deep-seated organs, such as the liver, pancreas, and kidney.
AuNPs are cancer-selective chemo reagents because they induce cancer-specific cytotoxicity. However, enhancement of cancer-specific accumulation is desired to achieve amore effective treatment. To obtain effective cancer-specific accumulation of AuNPs, we
focused on photodynamic therapy (PDT), a type of cancer treatment that is influenced
by the accumulation of photosensitizers, such as porphyrins [5]. Porphyrin accumulation
was shown to be higher in cancer tissues than in normal tissues. When exposed to laser
irradiation with the optimal wavelength, porphyrins induce the production of ROS via a
photoreaction, causing cancer-specific damage. Cellular uptake of porphyrin is induced byheme carrier protein-1 (HCP-1), whose expression is higher in cancer cells than in normal
cells [6,7]. We reasoned that porphyrin attachment could function in the accumulation of
AuNPs in cancer cells via HCP-1.
In this study, we synthesized a porphyrin-attached Au compound and found that thiscompound preferentially accumulates in cancer cells and induces cancer-specific cytotoxicity in combination with laser irradiation to generate the plasmonic effect.
2. Results
2.1. Preparation and Characterization of Au-HpD
HpD-PEG-SH and FL-PEG-SH were synthesized, and the porphyrin modification in
the Au compound, Au-HpD, was prepared by HpD-PEG-SH and FL-PEG-SH (Figure 1).
The introduction of HpD into HpD-PEG-SH was confirmed by gel permeation chromatography (GPC) (Figure S1). The elution of polymers with/without fluorescent dye, suchas HpD and FL, can be detected by the refractive index detector, while the elution offluorescent-labeled polymers can be detected by a UV detector. As shown in Figure S1(Supplementary Materials), heterofunctional polyethylene glycol with amine and thiolends (NH2-PEG-SH) had no absorbance, while HpD-PEG-SH showed strong absorbance at
400 nm at the elution time of HpD-PEG-SH. This result indicates the introduction of HpD
into the HpD-PEG-SH. From the GPC chromatogram, the peak of HpD-PEG-S-S-PEG-HpD
with a disulfide bond was detected. Therefore, tris (2-carboxyethyl) phosphine solution
was used for the reduction of the disulfide bond in the preparation of Au-HpD. In addition,
free HpD was detected in Figure S1B. Free HpD can be removed in the purification stepof Au-HpD. Similarly, the introduction of FL into the FL-PEG-SH was confirmed by GPC(Figure S2). The mean hydrodynamic diameter of Au-HpD was 58.8 nm, and the polydispersity index was 0.185 (Figure 2A). The UV–Vis absorption spectra in PBS are shown in
Figure 2B. Au has two bands at 530 nm and 870 nm. Au-HpD also had the same two bands.
Thus, the bands of Au-HpD were derived from Au.
2.2. Accumulation of Au-HpD Is Higher in Cancer Cells Than in Normal Cells
Taking into consideration that cancer cells incorporate higher levels of porphyrins
than normal cells, we analyzed whether Au-HpD has any preferential accumulation incancer cells. For this, RGK1 and RGM1 cells were incubated in a growth medium with
Au-HpD for 24 h. The analysis showed that Au-HpD absorption in RGK1 cancer cells
was significantly higher than in RGM1 normal cells (Figure 3). This result suggests that
intracellular Au-HpD accumulation in cancer cells was higher than in normal cells.
2.3. Au-HpD Induces Cytotoxicity in Cancer Cells
In order to analyze the cytotoxicity induced by Au-HpD, RGK-KO, and RGM-GFP,
cells were co-cultured in the presence of Au-HpD for 24 h and then subjected to 870 nm
laser irradiation for 30 min. RGM-GFP cells resulted from the transfection of RGM1 cellswith a construct expressing a green fluorescent protein (GFP) gene, which fluoresces ingreen when excited with blue light. RGK-KO cells were established by the transfection of
RGK1 cells with a construct expressing the kusabira orange (KO) protein gene [8]. In theentire cell population, RGK cancer cells were distinguishable from RGM normal cells as

Molecules 2022, 27, 1238

Molecules 2022, 27, 1238

Molecules 2022, 27, 1238

3 of 10they express different fluorescent proteins. We observed that, after laser irradiation, RGKcancer cells showed significant morphological changes, such as chromatin condensationand nuclear fragmentation indicative of cell apoptosis (Figure 4A,B,D,E). This strong effectwas not observed when RGM normal cells were irradiated (Figure 4A,C,D,F). Cell apoptosis
was detected using Hoechst 33342. The phase-contrast image is shown in Figure 5A, andthe fluorescence images are shown in Figure 5B–D. Morphological changes indicative ofcell apoptosis, such as condensation of both chromatin and nuclear fragmentation, wereobserved in RGK-KO cancer cells (Figure 5B,C). However, it was not observed in RGMGFP normal cells (Figure 5B,D). Taken together, our results show that there is increased
cytotoxicity associated with the amount of Au-HpD in the cell.

3

3 of 10Figureillustrationofporphyrin-modified
Au
Au-HpD.
FigureSchematic
illustrationof thetheofporphyrin-modified
Aucompound:compound:Au-HpD. Au-HpD.
Figure1.1.1.Schematic
Schematic
illustration
the porphyrin-modified
Au compound:Figure−Vis absorption
absorption
Figure2.
2. The
The characteristics
characteristics ofof Au-HpD:
Au-HpD: (A)(A) size
size distributions
distributions ofof Au-HpD;
Au-HpD; (B)(B) UVUV−Visspectrumof
ofAu-HpD
Au-HpDin
inaaPBS
PBSsolution.
solution.
spectrum

Figure 2. The characteristics of Au-HpD: (A) size distributions of Au-HpD; (B) UV−Vis absorp
2.2. Accumulation of Au-HpD Is Higher in Cancer Cells Than in Normal Cells
spectrum of Au-HpD in a PBS solution.
Taking into consideration that cancer cells incorporate higher levels of porphyrins
than normal cells, we analyzed whether Au-HpD has any preferential accumulation in
2.2. Accumulation of Au-HpD Is Higher in Cancer Cells Than in Normal Cells
cancer cells. For this, RGK1 and RGM1 cells were incubated in a growth medium withTakingconsideration
that
cells
incorporate
higherof porphy
Au-HpDfor 24intoh. Theanalysis showedthatcancer
Au-HpDabsorption
in RGK1
cancerlevels
cells wassignificantly
higherRGM1 normal
cells (Figure3). Thissuggests that intrathan normal
cells,thanweinanalyzed
whether
Au-HpDhas result
any preferential
accumulatio
cellular Au-HpD accumulation in cancer cells was higher than in normal cells.

Molecules 2022, 27, 1238

Molecules 2022, 27, 1238

4 of 10Figure 3. Intracellular Au-HpD accumulation in cancer cells and normal cells. Data are expressed
as means ± SD (n = 5). ** p < 0.01.

2.3. Au-HpD Induces Cytotoxicity in Cancer Cells

In order to analyze the cytotoxicity induced by Au-HpD, RGK-KO, and RGM-GFP,
cells were co-cultured in the presence of Au-HpD for 24 h and then subjected to 870 nm
laser irradiation for 30 min. RGM-GFP cells resulted from the transfection of RGM1 cellswith a construct expressing a green fluorescent protein (GFP) gene, which fluoresces ingreen when excited with blue light. RGK-KO cells were established by the transfection of
RGK1 cells with a construct expressing the kusabira orange (KO) protein gene [8]. In theentire cell population, RGK cancer cells were distinguishable from RGM normal cells as
they express different fluorescent proteins. We observed that, after laser irradiation, RGKcancer cells showed significant morphological changes, such as chromatin condensationand nuclear fragmentation indicative of cell apoptosis (Figure 4A,B,D,E). This strong effect was not observed when RGM normal cells were irradiated (Figure 4A,C,D,F). Cellapoptosis was detected using Hoechst 33342. The phase-contrast image is shown in Figure
5A, and the fluorescence images are shown in Figure 5B–D. Morphological changes indicative of cell apoptosis, such as condensation of both chromatin and nuclear fragmentation,
were observed in RGK-KO cancer cells (Figure 5B,C). However, it was not observed in
RGM-GFP
normal cells(Figure5B,D). Taken
together,
ournormal
results
show
thatis inFigure
Intracellular
Au-HpD
accumulationin cancer
cells
cells.
Data
are there
expressed
asData are
Figure3. 3.
Intracellular
Au-HpD
accumulation
inandcancercells
andnormalcells.
creasedcytotoxicity
associatedwiththeamountof
Au-HpDinthecell.
means ± SD (n = 5). ** p < 0.01.

as means ± SD (n = 5). ** p < 0.01.

2.3. Au-HpD Induces Cytotoxicity in Cancer Cells

In order to analyze the cytotoxicity induced by Au-HpD, RGK-KO, andcells were co-cultured in the presence of Au-HpD for 24 h and then subjected
laser irradiation for 30 min. RGM-GFP cells resulted from the transfection of R
with a construct expressing a green fluorescent protein (GFP) gene, which flgreen when excited with blue light. RGK-KO cells were established by the tran
RGK1 cells with a construct expressing the kusabira orange (KO) protein geneentire cell population, RGK cancer cells were distinguishable from RGM norm
they express different fluorescent proteins. We observed that, after laser irradi
cancer cells showed significant morphological changes, such as chromatin coand nuclear fragmentation indicative of cell apoptosis (Figure 4A,B,D,E). This
fect was not observed when RGM normal cells were irradiated (Figure 4A,C
Figure4.
combination
with
Au-HpD
and
irradiation
for
beforeapoptosis
was detectedusing
Hoechstphase-contrast
image
Figure4. TheThecytotoxicity
cytotoxicity
combination
with
Au-HpD33342.
andlaserlaserThe
irradiation
forcells:
cells:top
toprow,row,
beforeis show
laser irradiation; bottom row, after laser irradiation; phase-contrast image (A,D); fluorescence image
laser
bottom row, after laser
irradiation;phase-contrast
image (A,D);
fluorescence
image
5A, irradiation;and the fluorescence
imagesare shownin Figure
5B–D.
Morphologicalcha
of RGK-KO (B,E); fluorescence image of RGM-GFP (C,F).
of
RGK-KO(B,E);
fluorescence
image
of
RGM-GFP(C,F).
ative of cell apoptosis, such as condensation of both chromatin and nuclear fragwereobserved
RGK-KO
cancercellsCells(Figure5B,C). with
However,
it was not o
2.4.
Intracellular
ROSinGeneration
Increasedin RGKin Combination
Au-HpD and
Laser
Irradiation
RGM-GFP normal cells (Figure 5B,D). Taken together, our results show that
We analyzed
the productionof intracellularin cells of
with
Au-HpD.in
Wethe
observedcreasedcytotoxicity
associatedwith theROS
amountAu-HpD
cell.
that incubation with Au-HpD induced a significant increase in ROS in RGK1 cells, even
before they were laser irradiated, when compared to that in RGM1 normal cells. This result
indicates that intracellular ROS production in cancer cells treated with Au-HpD is higher
than in normal cells treated with the same compound. After irradiation, intracellular ROS
increased significantly in RGK1 cancer cells when compared with that in non-irradiatedcells. The intracellular ROS levels of RGM1 normal cells treated with Au-HpD were notsignificantly altered after irradiation (Figure 6).

Molecules
Molecules2022,
2022,27,
27, 1238
1238

10
5 5ofof10

Figure 5. Hoechst 33258 staining of RGK-KO and RGM-GFP cells: phase-contrast image (A); fluorescence image of Hoechst 33342 (B); RGK-KO (C); RGM-GFP (D).

2.4. Intracellular ROS Generation Increased in RGK Cells in Combination with Au-HpD and
Laser Irradiation
We analyzed the production of intracellular ROS in cells with Au-HpD. We observedthat incubation with Au-HpD induced a significant increase in ROS in RGK1 cells, even
before they were laser irradiated, when compared to that in RGM1 normal cells. This result indicates that intracellular ROS production in cancer cells treated with Au-HpD ishigher than in normal cells treated with the same compound. After irradiation, intracellular ROS increased significantly in RGK1 cancer cells when compared with that in nonFigure 5.
5. Hoechst
33258 staining
staining ofof RGK-KO
RGK-KO andand RGM-GFP cells:
cells: phase-contrast
phase-contrast image
image (A); fluoresfluoFigure
Hoechstirradiatedcells. 33258
The intracellular
ROS levels RGM-GFP
of RGM1 normal
cells treated with(A);Au-HpD
rescence image of Hoechst 33342 (B); RGK-KO (C); RGM-GFP (D).
cenceof Hoechst 33342(B);after
RGK-KO(C); RGM-GFP
wereimage
not significantly
alteredirradiation(Figure (D).
6).

2.4. Intracellular ROS Generation Increased in RGK Cells in Combination with Au-HpD and
Laser Irradiation
We analyzed the production of intracellular ROS in cells with Au-HpD. We observedthat incubation with Au-HpD induced a significant increase in ROS in RGK1 cells, even
before they were laser irradiated, when compared to that in RGM1 normal cells. This result indicates that intracellular ROS production in cancer cells treated with Au-HpD ishigher than in normal cells treated with the same compound. After irradiation, intracellular ROS increased significantly in RGK1 cancer cells when compared with that in nonirradiated cells. The intracellular ROS levels of RGM1 normal cells treated with Au-HpD
were not significantly altered after irradiation (Figure 6).

Figure 6. The fluorescence intensity of HPF: (A) fluorescent microscopy utilized to assess cellular
uptake of HPF; (B) data are expressed as means ± SD (n = 20). Ex = 460–495 nm and Em = 510–550 nm.
* p < 0.05, ** p < 0.01.

3. Discussion
In this study, we prepared Au-HpD and evaluated its characteristics. Intracellular
Au-HpD accumulation in RGK1 cells was significantly higher than in RGM1 normal cells.
Moreover, combination treatment with Au-HpD and laser irradiation induced cancerspecific apoptosis and ROS generation.
Nanoparticles have been developed with potential applications as diagnostic andtherapeutic agents, and nanoparticle-based therapies are a promising approach to overcome

Molecules 2022, 27, 1238

6 of 10conventional cancer therapies [8]. Magnetic nanoparticles can induce hyperthermia in thepresence of an alternating magnetic field [9,10]. In addition, metallic nanoparticles witha high atomic number release Auger electrons in response to X-ray radiation, having thepotential to locally enhance the effects of radiation therapy when specifically delivered totumors [11]. Over the past decades, therapy using AuNPs has been investigated for several
diseases, in particular for cancer [12,13]. However, the identification of modifications toachieve cancer-specific delivery is essential to induce more cancer-specific damage withlower effects in normal cells.
In this study, we focused on AuNPs, which have high biocompatibility and absorption
in the NIR region. The absorption coefficients of water and hemoglobin are at their lowest
levels in the NIR window, enabling light with longer wavelengths to deeply penetrate
the tissue [14]. The use of AuNPs with high cancer-specific accumulation allows forcancer treatment in deep tissues. To enhance the cancer-specific accumulation, we selected
porphyrins, which are used as photosensitizers in PDT and were shown to accumulate incancer cells. They can be activated by optimal wavelength laser irradiation, establishinginteractions with the surroundings via different pathways [15]. Such interactions generate
ROS that, in turn, induce cytotoxicity more prominently in tumor tissue due to cancerspecific porphyrin accumulation.
Au-HpD showed absorbance at 530 nm and 870 nm (Figure 3). We measured the
Au-HpD absorption at 870 nm in both rat gastric normal cells and the corresponding cancer
cells. Au-HpD accumulation in cancer cells was higher than in normal cells. Au-HpD
accumulation was higher than AuNPs (Figure S3). According to the porphyrin attachment,
Au-HpD obtained the cancer-specific accumulation.
Considering that ROS are formed when AuNPs are irradiated by an optimal laser
wavelength [16,17], we evaluated the cancer-specific cytotoxicity induced by the combination of Au-HpD and laser irradiation. To address this, we co-cultured normal cells andcancer cells and subjected them to the same conditions, recapitulating the tumor treatment
conditions. We observed that, after incorporation of Au-HpD, cancer cells that were laser
irradiated showed significant morphological alterations indicative of apoptosis. In parallel, normal cells under the same conditions did not show any morphological alterations.
These results suggest that the combination treatment (Au-HpD + laser irradiation) induced
cancer-specific apoptosis. Although cancer cells and normal cells were very close in the
co-culture system, the combination therapy could induce cytotoxicity specifically in cancer
cells, which is an encouraging result, with lower side effects in normal tissues. Moreover,this combination therapy used an emission light with an 870 nm wavelength, which canbe highly effective in the treatment of cancers in deeper regions of the organism. We also
attached the fluorescein in Au-HpD. After Au-HpD exposure and laser irradiation, if cancer
cells have no damage, we considered that laser power was too low or that Au-HpD accumulation was not enough. To confirm that Au-HpD accumulated in cancer cells, we attached
the fluorescein in Au-HpD. However, after Au-HpD accumulation and laser irradiation,
cancer cells showed cytotoxicity. Thus, we did not need confirmation using fluorescein.
Finally, we evaluated intracellular ROS production by the combination therapy. It
is already known that intracellular ROS production in cancer cells is higher than that innormal cells [18,19]. AuNPs can induce apoptosis through ROS production [20], and incombination with laser irradiation, they enhance ROS generation via localized surface
plasmon resonance [21]. In fact, in our experimental conditions, intracellular ROS production in cancer cells treated with Au-HpD was significantly higher than in normal cells
under the same treatment, even before laser irradiation (Figure 6). After laser irradiation,
intracellular ROS production in cancer cells increased, while intracellular ROS production
in normal cells remained virtually the same as before laser irradiation. AuNPs enhance
ROS generation by thermal effects or localized surface plasmon resonance. Au-HpD was
excited by NIR light, and thermal or plasmon resonance can enhance ROS production incancer cells. We discovered that the combination of Au-HpD and laser irradiation induced
cancer-specific cytotoxicity and ROS production.

Molecules 2022, 27, 1238

7 of 10

4. Materials and Methods
4.1. Reagents
AuNPs were purchased from Cytodiagnostics Inc. (Burlington, ON, Canada). HpD
was purchased from MedChem Express (San Diego, CA, USA). Fluorescein isothiocyanate(FITC) was purchased from Dojindo (Kumamoto, Japan). A mono-functional polyethylene glycol with thiol end (PEG-SH) and a heterofunctional polyethylene glycol withamine and thiol ends (Mw 2000) were purchased from NOF Corporation (Tokyo, Japan).
Dichloromethane (DCM), N,N-dimethylformamide (DMF), N-hydroxysuccinimide (NHS),
and N,N0 -diisopropylcarbodiimide were purchased from Fujifilm Wako Pure Chemical(Osaka, Japan) and used with no further purification.
4.2. Synthesis of HpD-PEG-SH
Heterofunctional polyethylene glycol with amine and thiol ends (100 mg) was weighed
into a 10 mL flask. A mixture of 2 mL of DCM and 2 mL of DMF containing N,N0 diisopropylcarbodiimide (126 mg), NHS (115 mg), triethylamine (20 mg), and HpD (33 mg)
was added to the flask and stirred for 2 h at 25 ◦ C. The HpD-PEG-SH was recovered by
precipitation into 40 mL of cold 2-propanol (−15 ◦ C) and centrifugation for 10 min at
5000 rpm (4500× g). The resulting PMETAC-co-PAPMAA copolymer was transferred intoa pre-swollen membrane tube (Spectra/Por; molecular-weight cutoff size: 1000), dialyzed
for 24 h against 2 L of water, which was changed after 2, 5, and 8 h, and then freeze-dried.
4.3. Synthesis of FL-PEG-SH
Heterofunctional polyethylene glycol with amine and thiol ends (100 mg) was weighed
into a 10 mL flask. A mixture of 2 mL of DMF containing triethylamine (20 mg) and FITC
(38 mg) was added to the flask and stirred for 2 h at 25 ◦ C. The FL-PEG-SH was recoveredby precipitation into 40 mL of cold 2-propanol (−15 ◦ C) and centrifugation for 10 min at
5000 rpm (4500× g). The resulting PMETAC-co-PAPMAA copolymer was transferred intoa pre-swollen membrane tube (Spectra/Por; molecular-weight cutoff size: 1000), dialyzed
for 24 h against 2 L water, which was changed after 2, 5, and 8 h, and then freeze-dried.
4.4. Preparation of Au-HpD
A volume of 10 µL of tris (2-carboxyethyl) phosphine solution (50 mg/mL) was added
to 300 µL of PEG stock solution containing 445 µg/mL of PEG-SH, 50 µg/mL of HpD-PEGSH, and 5 µg/mL of FL-PEG-SH to reduce disulfide bonds. The molar ratio of PEG-SH,
HpD-PEG-SH, and FL-PEG-SH was 107: 9:1. This mixture was allowed to react at 25 ◦ C
for 30 min and added to 75 µL of AuNPs solution. This mixture was allowed to react at
25 ◦ C for 1 h, followed by centrifugation at 15,000 rpm for 20 min at 20 ◦ C. The obtained
solution of AuNPs was centrifuged at 15,000× g for 20 min, decanted, and resuspended
in PBS to remove excess PEG derivatives. Figure 1 shows a schematic illustration of the
Au-HpD. The hydrodynamic diameters of Au-HpD were determined using a Zetasizer
Nano (Zetasizer Nanoseries ZEN3600; Malvern Instruments Ltd., Worcestershire, UK).
The absorbance of Au-HpD was measured using a Synergy H1 microplate reader (BioTek
Instruments Inc., Winooski, VT, USA).
4.5. Cell Culture
RGM1 rat gastric mucosal cells were purchased from the RIKEN Cell Bank (Tsukuba,
Japan). RGK1 cells were established by exposing RGM1 cells to 1-Methyl-3-nitro-1-nitrosoguanidine. RGM-GFP cells resulted from the transfection of RGM1 cells with a constructexpressing a green fluorescent protein (GFP) gene, which fluoresces in green when excited with blue light. RGK-KO cells were established by the transfection of RGK1 cellswith a construct expressing the kusabira orange (KO) protein gene [8]. RGM cells were
cultured in Dulbecco’s modified Eagles/F12 medium (Life Technologies Japan, Tokyo,
Japan). RGK cells were cultured in Dulbecco’s modified Eagles/F12 medium without
L-glutamine (Sigma-Aldrich Japan K.K., Tokyo, Japan). These culture media containedMolecules 2022, 27, 1238

8 of 10

10% heat-inactivated fetal bovine serum (Biowest, Kansas City, MO, USA) and 1% penicillin/streptomycin (Wako Pure Chemical Industries, Osaka, Japan). Cells were cultured ina 37 ◦ C incubator in an atmosphere of 5% CO2 .
4.6. Cellular Uptake of Au-HpD
RGK1 and RGM1 cells were seeded in 12-well plates at a density of 5 × 104 cells/welland incubated at 37 ◦ C for 24 h. Au-HpD was added to cells, and 24 h later, cells wererinsed with phosphate-buffered solution (PBS) and lysed in 100 µL of RIPA buffer. The
cell homogenates were transferred to a 96-well plate, and the absorbance was measured
at 870 nm using a Synergy H1 microplate reader (BioTek Instruments Inc., Winooski, VT,
USA). The protein concentration of each sample was estimated by the bicinchoninic acidprotein assay kit (Takara Bio, Shiga, Japan). The absorbance was measured at 562 nm usinga Synergy H1 microplate reader.
4.7. PDT Using Au-HpD
RGK1 and RGM1 cells were grown overnight in a 60 mm dish and then incubatedwith Au-HpD for 24 h. After incubation, the medium was changed to fresh medium
without phenol red, and cells were irradiated with supercontinuum laser light (870 nm,
1.5 ± 3 mW/cm2 ) using a SuperK FIANIUM FIU-15 (NKT Photonics, Birkerod, Denmark).
4.8. Detection of Apoptotic Cells by Hoechst 33258 Staining
RGK1 and RGM1 cells were cultured in 60 mm dishes and incubated overnight. After
PDT treatment, RGK1 and RGM1 cells were incubated with 5 µM Hoechst 33342 for 30 min.
Apoptosis, with condensed and fragmented nuclei, was analyzed using the fluorescencemicroscope IX83.
4.9. Intracellular Reactive Oxygen Species (ROS) Measurement
Intracellular ROS were detected using a 2-[6-(4-hydroxy) phenoxy-3H-xanthen-3on-9-yl] benzoic acid (HPF) (Goryo Chemical, Hokkaido, Japan) fluorescence dye. Nonfluorescent HPF would be O-dearylated upon reaction with ROS and then show strong
fluorescence. RGK1 and RGM1 cells were cultured in 60 mm dishes overnight. After PDT
treatment, RGK1 and RGM1 cells were incubated with 5 µM HPF in MSF buffer (5.4 mM
KCl, 136.9 mM NaCl, 8.3 mM glucose, 0.44 mM KH2 PO4 , 0.33 Na2 HPO4 , 10.1 mM HEPES,
1 mM MgCl6 6H2 O, 1 mM CaCl2 2H2 O) for 10 min. After incubation, the solution was
replaced with fresh MSF buffer. Fluorescence intensity was analyzed using a fluorescencemicroscope IX83 (Olympus Optical Co Ltd., Tokyo, Japan). HPF was excited using a
460–495 nm filter, and the emission was collected using a 510–550 nm filter. The protein
concentration of each sample was estimated by the bicinchoninic acid protein assay kit(Takara Bio, Shiga, Japan). The absorbance was measured at 562 nm using a Synergy H1
microplate reader.
4.10. Statistical Analysis
Data are expressed as the means ± SD and were assessed by an analysis of variance.
Individual groups were compared by Tukey’s post hoc or Student’s t-test with p < 0.05
considered statistically significant.
5. Conclusions
In this study, we describe the use of Au nanoparticles fused with porphyrins in a
combination therapy with NIR laser treatment to induce cytotoxicity specifically in cancer
and not in normal cells in the same culture. These are encouraging results for the use ofthis combination therapy in the treatment of different tumors. Additionally, the use of NIR
light is expected to induce cancer-specific cell death in deep regions of the organism.

Molecules 2022, 27, 1238

9 of 10Supplementary Materials: The following are available online. Figure S1: Gel permeation chromatograms of NH2-PEG-SH and HpD-PEG-SH; Figure S2: Gel permeation chromatograms of NH2PEG-SH and FL-PEG-SH; Figure S3: Intracellular AuNPs and Au-HpD accumulation in cancer cells.
Author Contributions: H.K., A.T., T.Y., H.S. and H.M. designed the experiments. H.K., A.T. and T.Y.
performed and analyzed the experiments. All authors have read and agreed to the published versionof the manuscript.
Funding: This study was partially supported by JSPS Grant-in-Aid for Scientific Research JP17H06088,
JP19H02591, and JP19K16854 and CREST (JPMJCR1875).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.

References
1.
2.
3.

4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

Mattiuzzi, C.; Lippi, G. Current cancer epidemiology. J. Epidemiol. Glob. Health 2019, 9, 217–222. [CrossRef] [PubMed]
Daly, L.E.; Dolan, R.D.; Power, D.G.; Rd, N.B.; Sim, W.; Rd, S.J.C.; Fallon, M.; Simmons, C.; McMillan, D.C.; Rd, A.M.R.; et al.
Determinants of quality of life in patients with incurable cancer. Cancer 2020, 126, 2872–2882. [CrossRef] [PubMed]
Mamalis, A.; Koo, E.; Sckisel, G.D.; Siegel, D.M.; Jagdeo, J. Temperature-dependent impact of thermal aminolaevulinic acidphotodynamic therapy on apoptosis and reactive oxygen species generation in human dermal fibroblasts. Br. J. Dermatol. 2016,
175, 512–519. [CrossRef] [PubMed]
Zhang, Z.; Wang, S.; Xu, H.; Wang, B.; Yao, C. Role of 5-aminolevulinic acid-conjugated gold nanoparticles for photodynamic
therapy of cancer. J. Biomed. Opt. 2015, 20, 051043. [CrossRef]
Abrahamse, H.; Hamblin, M.R. New photosensitizers for photodynamic therapy. Biochem. J. 2016, 473, 347–364. [CrossRef]
Ito, H.; Matsui, H.; Tamura, M.; Majima, H.J.; Indo, H.P.; Hyodo, I. Mitochondrial reactive oxygen species accelerate the expressionof heme carrier protein 1 and enhance photodynamic cancer therapy effect. J. Clin. Biochem. Nutr. 2014, 55, 67–71. [CrossRef]
Hiyama, K.; Matsui, H.; Tamura, M.; Shimokawa, O.; Hiyama, M.; Kaneko, T.; Nagano, Y.; Hyodo, I.; Tanaka, J.; Miwa, Y.; et al.
Cancer cells uptake porphyrins via heme carrier protein 1. J. Porphyr. Phthalocyanines 2013, 17, 36–43. [CrossRef]
Baetke, S.C.; Lammers, T.; Kiessling, F. Applications of nanoparticles for diagnosis and therapy of cancer. Br. J. Radiol. 2015, 88,1054. [CrossRef]
Yen, S.K.; Padmanabhan, P.; Selvan, S.T. Multifunctional Iron Oxide Nanoparticles for Diagnostics, Therapy and Macromolecule
Delivery. Theranostics 2013, 3, 986–1003. [CrossRef]
Quinto, C.A.; Mohindra, P.; Tong, S.; Bao, G. Multifunctional superparamagnetic iron oxide nanoparticles for combined
chemotherapy and hyperthermia cancer treatment. Nanoscale 2015, 7, 12728–12736. [CrossRef]
Davidson, R.A.; Guo, T. Nanoparticle-Assisted Scanning Focusing X-Ray Therapy with Needle Beam X rays. Radiat. Res. 2016,
185, 87–95. [CrossRef] [PubMed]
Boisselier, E.; Astruc, D. Gold nanoparticles in nanomedicine: Preparations, imaging, diagnostics, therapies and toxicity. Chem.
Soc. Rev. 2009, 38, 1759–1782. [CrossRef] [PubMed]
Yu, Y.; Yang, T.; Sun, T. New insights into the synthesis, toxicity and applications of gold nanoparticles in CT imaging andtreatment of cancer. Nanomedicine 2020, 15, 1127–1145. [CrossRef] [PubMed]
Sordillo, L.A.; Pu, Y.; Pratavieira, S.; Budansky, Y.; Alfano, R.R. Deep optical imaging of tissue using the second and thirdnear-infrared spectral windows. J. Biomed. Opt. 2014, 19, 056004. [CrossRef] [PubMed]
Calixto, G.M.; Bernegossi, J.; de Freitas, L.M.; Fontana, C.R.; Chorilli, M. Nanotechnology-based drug delivery systems forphotodynamic therapy of cancer: A review. Molecules 2016, 21, 342. [CrossRef] [PubMed]
Pasparakis, G. Light-Induced Generation of Singlet Oxygen by Naked Gold Nanoparticles and its Implications to Cancer Cell
Phototherapy. Small 2013, 9, 4130–4134. [CrossRef]
Yoo, D.; Jeong, H.; Preihs, C.; Choi, J.S.; Shin, T.H.; Sessler, J.L.; Cheon, J. Double-effector nanoparticles: A synergistic approach toapoptotic hyperthermia. Angew. Chem. (Int. Ed. Engl.) 2012, 51, 12482–12485. [CrossRef]
Sullivan, L.B.; Chandel, N.S. Mitochondrial reactive oxygen species and cancer. Cancer Metab. 2014, 2, 17. [CrossRef]
Tamura, M.; Matsui, H.; Tomita, T.; Sadakata, H.; Indo, H.P.; Majima, H.J.; Kaneko, T.; Hyodo, I. Mitochondrial reactive oxygen
species accelerate gastric cancer cell invasion. J. Clin. Biochem. Nutr. 2014, 54, 12–17. [CrossRef]

Molecules 2022, 27, 1238

20.

21.

10 of 10Chompoosor, A.; Saha, K.; Ghosh, P.S.; Macarthy, D.J.; Miranda, O.R.; Zhu, Z.-J.; Arcaro, K.F.; Rotello, V.M. The Role of Surface
Functionality on Acute Cytotoxicity, ROS Generation and DNA Damage by Cationic Gold Nanoparticles. Small 2010, 6, 2246–2249.
[CrossRef]
Zhang, D.; Lin, X.; Lan, S.; Sun, H.; Wang, X.; Liu, X.; Zhang, Y.; Zeng, Y. Localized surface plasmon resonance enhanced singlet
oxygen generation and light absorption based on black phosphorus@aunps nanosheet for tumor photodynamic/thermal therapy.
Part. Part. Syst. Charact. 2018, 35, 1800010. [CrossRef]