Materials
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ISSN 2633-5409

PAPER
Teruyuki Komatsu et al.
Catalase–albumin cluster incorporating protoporphyrin
IX: an O2 generating photosensitizer for enhanced
photodynamic therapy

Volume 3
Number 16
21 August 2022
Pages 6387–6658

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3, 6451

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Catalase–albumin cluster incorporating
protoporphyrin IX: an O2 generating
photosensitizer for enhanced photodynamic
therapy†
Taiga Yamada, Maho Katsumi, Yuka Yagisawa, Masato Ichihara andTeruyuki Komatsu *Photodynamic therapy (PDT) is a non-invasive cancer treatment using reactive oxygen species (ROS)
generated by light irradiation. An excited state photosensitizer produces active radicals (e.g. O2

andOH ) by electron transfer reactions (type I), while an excited triplet state photosensitizer generates
singlet O2 (1O2) by direct energy transfer to molecular O2 (type II). This report describes the synthesis
and PDT activity of the hemoprotein-based photosensitizer combined with O2 production capability.
Covalent wrapping of a catalase (Cat) with human serum albumins (HSAs) yielded the core–shell structured protein cluster Cat-HSA5, in which an average of five HSA molecules are bound to the Cat center.
Cat-HSA5 retained the high enzymatic activity of the hydrogen peroxide (H2O2) disproportionation
reaction. Subsequently, the incorporation of protoporphyrin IX (PP) into an individual HSA unit resulted
in a Cat-(HSA-PP)5 cluster. The 1O2 generation ability of Cat-(HSA-PP)5 was equivalent to that of theReceived 2nd March 2022,
Accepted 17th June 2022

HSA–PP complex. Cytotoxicity with light irradiation was evaluated using HeLa cells. Cat-(HSA-PP)5
demonstrated higher PDT activity than the HSA–PP complex. The Cat core provoked conversion of

DOI: 10.1039/d2ma00242f

endogenous H2O2 to O2 efficiently in the cells to facilitate 1O2 formation in large amounts. Cat-(HSA-

rsc.li/materials-advancesPP)5 is a unique dual functional hemoprotein cluster (O2 generation and photosensitization) that canachieve enhanced PDT.

Department of Applied Chemistry, Faculty of Science and Engineering, Chuo
University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan.
E-mail: komatsu@kc.chuo-u.ac.jp† Electronic supplementary information (ESI) available: Native-PAGE, SDS-PAGE
and DLS of Cat-HSA5, Lineweaver–Burk plots of H2O2 degradation using CatHSA5, time course of O2 generated by H2O2 dismutation using Cat-HSA5, absorption and fluorescence spectra of HSA-PP, fluorescence, transient absorption
spectra and time course of the fluorescence intensity of Cat-(HSA-PP)5, and cell
viability of HeLa cells treated with Cat-(HSA-PP)5 without or with light irradiation.
See DOI: https://doi.org/10.1039/d2ma00242f(50–100 mM),7 and (iii) in situ O2-production by the splittingof H2O.8 Cheng et al. reported that the perfluorocarbon-based
O2-carrier including IR780 facilitates photodynamic activity.6
Tae et al. demonstrated that graphene oxide with hemin andchlorin e6 becomes a useful reagent for PDT.9 Photosensitizers
must accumulate in the affected part. Generally, nanoparticles
can access the tumor tissue closely by enhanced permeability
and retention (EPR) effects.10,11 Yan et al. reported that carrier
free self-assembled nanoparticles based on chlorine e6 showedsuperior anticancer activity.12 Using EPR effects, one can makenanoparticles to penetrate gaps in tumor blood vessels and todiffuse in the tissues (passive targeting).13 Accumulation intumors by passive targeting is, however, only 20–30% superior
to that observed in normal tissues. Another technique is to use
receptors that are overexpressed on tumors.13 Immobilizationof receptor binding ligands on the nanoparticle conferred highaffinity with tumor tissues to the particles (active targeting).13
For example, RGD peptides (arginylglycylaspartic acid) bindaVb3 integrin on the vascular endothelial cell in the TME, whichcauses enhancement of cellular uptake.14
Earlier, we reported that Hb-HSA3, a core–shell protein
cluster comprising hemoglobin (Hb) and human serum© 2022 The Author(s). Published by the Royal Society of Chemistry

Mater. Adv., 2022, 3, 6451–6457 | 6451

Introduction
Photodynamic therapy (PDT) is a non-surgical cancer treatmentusing a light source such as an LED or laser.1–3 A singlet oxygen(1O2) generated by energy transfer from a photosensitizing
agent to O2 is implicated as an intermediary species leading
to cell death. However, hypoxia (pO2 o 2.5 Torr) in a tumor
microenvironment (TME)4 inhibits the 1O2 formation andthereby restricts PDT.5 Some investigators have reported theimprovement of hypoxia in the TME using different means: (i)direct O2-transportation using an O2 carrier,6 (ii) in situ O2generation by the disproportionation of endogenous H2O2

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Materials Advancesalbumins (HSAs), showed long circulation lifetime in the bloodstream as an artificial O2 carrier.15–17 Several animal experiments have clarified their safety and efficacy as a red blood cell
substitute.18–20 The peripheral HSA units of Hb-HSA3 can binda wide range of hydrophobic compounds and drugs.21 In fact,
the incorporation of protoporphyrin IX (PP) into Hb-HSA3 [Hb(HSA-PP0.7)3] allowed adequate PDT activity to this cluster.22
Furthermore, the Hb core brings O2 to the cell interior toaccelerate the photodynamic properties. To our surprise,
metHb-(HSA-PP0.7)3 with a ferric Hb center showed higher
PDT performance than the ferrous Hb-(HSA-PP0.7)3.22 One
possible explanation is that the H2O2 disproportionation reaction by the metHb core improved hypoxia more efficiently thanthe O2-transport by the Hb core. It is known that metHb
exhibits weak peroxidase and catalase activities.23 To confer
stronger O2 generation ability to the cluster, a catalase (Cat),
which is the most powerful H2O2 dismutation enzyme innature, should be exploited instead of a metHb core. For thisstudy, we have demonstrated the synthesis, structure, andenzymatic activity of the Cat-HSA5 cluster (Fig. 1). Moreover,the photophysical properties and PDT activity of Cat-HSA5
incorporating PP [Cat-(HSA-PP)5, Fig. 1] have been characterized. The results indicate that our approach can create a newclass of dual-functional hemoprotein-based medicines foradvanced PDT.

Results and discussion
Synthesis of the Cat-HSA5 cluster
Covalent wrapping of Cat with HSAs was achieved using abifunctional cross-linking agent, N-succinimidyl 3-maleimidopropionate (SMP), which selectively connects a primary amineof Lys and sulfhydryl group of Cys (Fig. 1).17 First, a total of 16
free Cys residues of Cat were blocked by N-ethyl maleimide(NEM).24 The obtained Cat was reacted with SMP to prepare
maleimide-activated Cat (MA-Cat). Second, the maleimide
terminals of MA-Cat were coupled site-specifically with a Cys34 of HSA. The cluster formation was confirmed using size
exclusion chromatography (SEC) (Fig. 2(A)). The reaction mixture exhibited a new broad peak at the high molecular weightFig. 2 (A) SEC profiles of the reaction mixture, Cat, and Cat-HSA5. (B) CD
spectra of Cat-HSA5, Cat, and HSA (1 mM) in PBS (pH 7.4) at 25 1C.

Fig. 1 Synthetic scheme of the Cat-HSA5 cluster and Cat-(HSA-PP)5
cluster.

region (14 min), which could be Cat-HSAm derivatives. The
unbound HSA was excluded using gel filtration chromatography (GPC) (yield: 70%) (Fig. 2(A)). The native-PAGE analysis of
Cat-HSAm showed multiple bands (Fig. S1A, ESI†), suggestingthat Cat-HSAm is a mixture of clusters with diﬀerent HSA
binding numbers. The SDS–PAGE analysis of Cat-HSAm also
showed new bands at 112 kDa corresponding to a Cat unit
(55 kDa) + 1HSA and at 176 kDa corresponding to a Cat unit +2HSA (Fig. S1B, ESI†). From all these results of SEC, nativePAGE, and SDS–PAGE, we concluded that the HSA wascovalently bound to the Cat core.
Based on assays of the total protein concentration and Cat
concentration, the HSA/Cat ratio was found to be
5.0 (mol mol 1). The CD spectral pattern and intensity of CatHSAm coincided closely with the sum of the Hb spectrum and afive-fold enlarged HSA spectrum (Fig. 2(B)). These results
indicated that (i) the average number of HSA units aroundthe Cat is 5, and that (ii) the secondary structure of theindividual protein component remains unaltered after the
HSA conjugation. We designated this catalase–albumin cluster
as Cat-HSA5. Subsequent DLS measurements showed that thediameter of Cat-HSA5 (17.6 nm) is 1.3-fold greater than that of

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Cat (13.6 nm) (Fig. S2, ESI†). A detailed inspection of the CatHSA5 structure using transmission electron microscopy andscanning electron microscopy unfortunately failed, because theparticle size of Cat-HSA5 was too small to analyze. In general,nanomaterials with a diameter of less than 100 nm exhibited
selective cytotoxicity because they penetrate tumor blood vessels as a result of the EPR effect.11 Moreover, a 60 kDa
glycoprotein (gp60) on the endothelial cell binds the HSA andtakes the protein into the tumor by transcytosis.25 Secreted
protein acidic and rich in Cys (SPARC) overexpressed on various
tumor tissues catches the HSA.26 For these reasons, we expectthat Cat-HSA5 binds strongly to the tumor cells and that it isaccumulated inside the cells. Apparently, the small particle size
(approx. 18 nm) and plasma protein wrapping might be beneficial for both the enhanced EPR effect and active targeting that
result in high accumulation in tumor tissues.
Enzyme activity of the Cat-HSA5 cluster
Cat decomposes H2O2 into O2 and H2O in a two-step reaction(2H2O2 - O2 + 2H2O).27 First, the active site of heme reacts
with H2O2, generating an oxoferryl porphyrin cation radical(Compound I) (eqn (1)). Subsequently, Compound I reacts withanother H2O2 to produce O2 (eqn (2)).
Cat + H2O2 - Compound I + H2O(1)

Compound I + H2O2 - Cat + O2 + H2O(2)Upon the addition of Cat-HSA5 into H2O2 solution, theabsorbance at 240 nm decreased immediately, signaling thedegradation of H2O2. The kcat/Km (kcat, catalytic constant; Km,
Michaelis constant) value of Cat-HSA5 was calculated using
Lineweaver–Burk plots (Fig. S3, ESI†) as 1.58  107 M 1 s 1,
which was comparable with the value of Cat (1.57  107 M 1 s 1).
The amount of generated O2 was also measured using a Clarktype O2 electrode. After the addition of H2O2 solution to theelectrode chamber, the dissolved O2 concentration increased
gradually. The velocity of the O2 generation was almost thesame as that of H2O2 degradation observed above. The rates ofO2 generation using Cat-HSA5 and Cat were almost equal ateach H2O2 concentration (0.5–4.0 mM) (Fig. S4, ESI†). The kcat/
Km value of Cat-HSA5 was 1.45  107 M 1 s 1, which closely
resembled that of Cat (1.49  107 M 1 s 1) and which agreed
well with that obtained from the H2O2 degradation. The
enzyme activity of the Cat core was retained after the HSA
binding. These observations suggest that the modification ofLys residues in Cat did not change the molecular environment
around the prosthetic heme group. This result is consistent
with the finding reported by Davis et al. that PEG-conjugated
Cat and Cat possess almost equal catalytic activities.28 We
presume that Cat-HSA5 can generate O2 more efficiently than
metHb-HSA3 by the dismutation reaction of endogenous H2O2
in TME because the catalytic activity of Cat-HSA5 is muchhigher than that of metHb-HSA3 (kcat/Km = 1.0  102 M 1 s 1).22© 2022 The Author(s). Published by the Royal Society of Chemistry

Photophysical properties of the Cat-(HSA-PP)5 cluster
The HSA captures various insoluble endogenous and exogenous compounds. Common drug binding sites 1 and 2 are
known to locate respectively in subdomains IIA and IIIA.21,29
Many dyes can be used as photosensitizers for PDT. The mostcommon currently used compounds are porphyrin derivatives.
PP, a natural pigment, is bound strongly (Ka: B105 M 1) to thenarrow D-shaped cavity in the subdomain IB of the HSA (drugbinding site 3) and shows distinct absorption bands including
a 630 nm band in the biological window.30 In contrast, theaqueous solution of PP demonstrated the broadened split Soretband absorption (lmax: 361, 441 nm) and no fluorescenceemission (Fig. S5, ESI†). This finding is attributable to the factthat PP molecules form J-aggregate assemblies in water.31
These characteristics diﬀer greatly from the typical spectral
shapes of the monomeric PP in the DMSO solution. It isnoteworthy that the absorption and fluorescence spectral patterns of the HSA–PP complex and PP in DMSO are fundamentally the same as their general features (Fig. S5, ESI†), which
implies that PP is monomolecularly bound within the HSA. The
difference absorption spectrum of Cat-(HSA-PP)5 minus CatHSA5 coincided perfectly with the Soret and Q bands of HSA–PP(Fig. 3). We inferred that the five PP molecules are accommodated in the individual HSA shells of Cat-HSA5. In the GFC
chromatogram of Cat-(HSA-PP)5, no peak of unbound PP wasfound. Consequently, Cat-(HSA-PP)5 was exploited without
further purification for the following photophysical and cytotoxicity experiments.
Cat-(HSA-PP)5 showed a strong fluorescence of PP at 699 nm(lex: 630 nm; Fig. S6A, ESI†). The intensity was slightly lower
than that of HSA–PP because Cat-HSA5 absorbs at 630 nm. The
fluorescence intensity of Cat-(HSA-PP)5 at 699 nm in PBS at
37 1C was almost unaltered during 7 days (Fig. S7, ESI†). Cat(HSA-PP)5 was sufficiently stable under physiological conditions. The transient absorption spectrum of Cat-(HSA-PP)5 at
200 ns after laser flash photolysis exhibited a positive band at

Fig. 3 Visible absorption diﬀerence spectrum of Cat-(HSA-PP)5 ([Cat] =4 mM) minus Cat-HSA5 ([Cat] = 4 mM) and comparison with that of HSA–PP([PP] = 20 mM) in PBS (pH 7.4) at 25 1C.

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Materials Advances

450 nm, which is based on the triplet–triplet (T–T) absorptionof PP (Fig. S6B, ESI†). Concomitantly, the ground state bleaching of PP was also observable at 410 nm. The time dependenceof the absorption decay was composed of a single-exponential
profile. The spectral pattern and triplet lifetime (tT = 0.96 ms)
were almost identical to those of HSA–PP (tT = 0.90 ms). Also,
Hb-(HSA-PP)3 and metHb-(HSA-PP)3 respectively showed similar tT values (0.87 ms and 0.92 ms).22 All these results impliedthat electron transfer did not occur from the excited state of PP
to the hemin groups of the Cat core.
Next, the 1O2 generation ability of Cat-(HSA-PP)5 was evaluated. The 1O2 formed by energy transfer from the excited
photosensitizer to O2 leads to apoptosis of cells. The 1O2
scavenging reagent, ABDA, was used to detect 1O2. Photoirradiation (l 4 620 nm) of the photosensitizer solution containing
ABDA caused bleaching at 380 nm, which is ascribed to ABDA
oxidation.32,33 This result supported the 1O2 formation by
excited Cat-(HSA-PP)5. The reaction rate using Cat-(HSA-PP)5
was almost identical to those obtained using HSA–PP, Hb-(HSAPP0.7)3, and metHb-(HSA-PP0.7)3 (Fig. 4).22 Because the tT valueand 1O2 generation ability of each photosensitizer were comparable, the PDT activity is expected to be proportional to theO2 production ability in tumors.
PDT activity of the Cat-(HSA-PP)5 cluster
Cytotoxicity and PDT activity were evaluated using HeLa cells.
Cat-(HSA-PP)5 and metHb-(HSA-PP0.7)3 can generate O2 by the
enzymatic disproportionation reaction of endogenous H2O2.
Although Hb-(HSA-PP0.7)3 can transport O2 directly into thecells, HSA–PP cannot supply O2. Without light irradiation, thecell viability of each group was always beyond 90% irrespectiveof the PP concentration (0–3 mM) (Fig. 5(A)). The results of thepresent study show that the cytotoxicity of the four protein
medicines was negligibly low under the dark condition.
Moreover, the cytotoxicities of Cat-(HSA-PP)5 and HSA-PP
were evaluated under a wide range of PP concentrations

Fig. 5 Cell viability of HeLa cells exposed to Cat-(HSA-PP)5, HSA-PP,
metHb-(HSA-PP0.7)3, and Hb-(HSA-PP0.7)3: (A) without light irradiation and(B) with light irradiation (l 4 620 nm, 25 mW cm 2 at 630 nm, 5 min). Eachvalue represents the mean  SD (n = 3). aRef. 22.

Fig. 4 Time courses of the residual ABDA percentage of Cat-(HSA-PP)5,
HSA-PP, Hb-(HSA-PP0.7)3, and metHb-(HSA-PP0.7)3 in PBS (pH 7.4) upon
exposure to a LED lamp with a filter (l 4 620 nm, 25 mW cm 2 at 630 nm).
aRef. 22.

([PP] = 0–50 mM). The cell viability of Cat-(HSA-PP)5 was slightly
higher than that of HSA–PP (Fig. S8, ESI†). The half maximal
inhibitory concentrations (IC50) of Cat-(HSA-PP)5 and HSA-PP
were ascertained as 47 mM and 36 mM, respectively. The cell
viability decreased markedly by photoirradiation, dependent on
the PP concentration (Fig. 5(B)). Microscopic observations of
the HeLa cells after trypan blue staining also showed that lightirradiation with Cat-(HSA-PP)5 definitely induced cell death(Fig. S9, ESI†). This result implied that 1O2 was generated in
the cells. Hb-(HSA-PP0.7)3 showed higher PDT activity than HSAPP arising from O2 delivery by the Hb core. The IC50 value ofHb-(HSA-PP0.7)3 was ascertained as 1.8 mM compared to 2.4 mM
of HSA–PP. It is particularly interesting that metHb-(HSAPP0.7)3 showed further high photodynamic activity in comparison to Hb-(HSA-PP0.7)3.22 The amount of generated O2 by the
H2O2 disproportionation reaction with metHb in the cells ispresumably greater than the transported O2 by Hb. As expected,
Cat-(HSA-PP)5 showed much greater PDT activity (IC50 =0.7 mM). In addition, we evaluated the PDT activities of6454 | Mater. Adv., 2022, 3, 6451–6457© 2022 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Materials AdvancesCat-(HSA-PP)5 and HSA–PP under hypoxic conditions ([O2] =1%). Although the cell viability was higher than that under
normoxic conditions ([O2] = 20%), Cat-(HSA-PP)5 showed superior PDT activity compared to HSA–PP (Fig. S10, ESI†). We
concluded that enzymatic O2 formation by H2O2 disproportionation in tumor cells was valid to promote PDT. Zhao et al.
reported that hyaluronic acid-based nano-particles (approx.
200 nm) including Cat and chlorin e6 were accumulated
efficiently in tumor tissues and that they acted as a good
photosensitizer.34 Hyaluronic acid is known to target the overexpressed CD44 receptor in several tumors. We expect that Cat(HSA-PP)5 with a diameter of approximately 18 nm and an HSA
envelope would be beneficial both for its EPR effect and its
active targeting to the tumor tissue in vivo.

Conclusions
Simple covalent binding of HSAs to a Cat provided a core–shellstructured Cat-HSA5 cluster. The protein assay and CD spectrum revealed that the average binding number of HSAs to Cat
was 5.0. The enzymatic activity of the Cat-HSA5 cluster wasmaintained after HSA wrapping. Addition of PP into the aqueous Cat-HSA5 solution enabled us to prepare the Cat-(HSA-PP)5
complex. UV-vis absorption, fluorescence emission, and T–T
absorption spectra proved that PPs were incorporated into thesubdomain IB of the HSA entities in Cat-HSA5. The tT and 1O2
generation ability of Cat-(HSA-PP)5 were comparable to those of
HSA–PP. As a consequence, Cat-(HSA-PP)5 exhibited markedly
high photodynamic activity compared to Hb-(HSA-PP0.7)3 andmetHb-(HSA-PP0.7)3. The superior H2O2 dismutation ability of
the Cat core unit to form O2 allowed the peripheral HSA–PP
photosensitizing units to enhance 1O2 production. Overall, ourresults have demonstrated Cat-(HSA-PP)5 as a promising hemoprotein cluster with dual functionalities that can be of tremendous clinical importance as an O2 generating photosensitizer.

Experimental section

Paper
SEC was conducted using a HPLC system (Prominence LC20AD/CTO-20A/SPD-20A; Shimadzu Corp.) with an SEC column(YMC-Pack Diol-300 S-5; YMC Co. Ltd) using 50 mM PB (pH 7.4)
as the mobile phase.
Synthesis of the Cat-HSA5 cluster
PBS solution (pH 7.4) of NEM (40 mM, 0.045 mL) was poured
into Cat solution (30 mM, 2 mL, PBS). The mixture (NEM/Cat =30, mol mol 1) was stirred under dark conditions for 1 hr at
25 1C. Then, the DMSO solution of SMP (18 mM, 0.2 mL) was
added to the reactant (SMP/Cat = 60, mol mol 1). After stirringfor 1 h, excess NEM and SMP were removed by gel filtration
chromatography (GFC) using a desalting column (PD-10 Sephadex G-25; Cytiva). The collected solution of maleimide-activatedCat (MA-Cat) was concentrated to 2 mL ([Cat] = 30 mM) using acentrifugal filter device (Amicon ultra-15, 10 kDa MWCO;
Merck KGaA) and was added gradually into the PBS solutionof HSA (1.8 mM, 2.0 mL). The resultant (HSA/MA-Cat =60, mol mol 1) was stirred gently under dark conditions for
18 hr at 4 1C. The obtained reaction mixture was subjected to
GFC with Superdex 200 pg (Cytiva) to remove the unboundexcess HSA. Based on the total protein assay using the protein
assay kit (Pierce 660 nm; Thermo Fisher Scientific Inc.)and Cat assay using the UV-vis absorption spectrum(e405 = 2.5  105 M 1 cm 1), the average HSA/Cat ratio of the
product was calculated as 5.0. The obtained catalase–albumincluster was designated as Cat-HSA5.
CD and DLS measurements
Circular dichroism (CD) spectra were obtained using a spectropolarimeter (J-820; Jasco Corp.). A quartz cuvette (1 mm optical
path length) was used for measurements of 200–250 nm. The
molecular size of the cluster was ascertained from dynamic
light scattering (DLS) measurements using a zetapotential andparticle size analyzer (ELSZ-2000ZS; Otsuka Electronics Co.,
Ltd). The diameters were estimated from the particle-size
distribution by the intensity. All measurements were taken at
25 1C.

Materials and apparatus

Enzyme activity of the Cat-HSA5 cluster

Bovine liver catalase (Cat), N-succinimidyl 3-maleimidopropionate (SMP), N-ethylmaleimide (NEM), antifoaming agent(Antifoam SI), hydrogen peroxide (H2O2), and 0.4 w/v% trypan
blue were purchased from Fujifilm Wako Pure Chemical Corp.
The human serum albumin (HSA) was purchased from Nihon
Pharmaceutical Co., Ltd. Protoporphyrin IX (PP) and 9,10anthracenediyl-bis(methylene) dimalonic acid (ABDA) were purchased from Merck KGaA. A Cell Counting Kit-8 (CCK-8) was
purchased from Dojindo Laboratories. Other chemical reagentsof special grade were used without further purification unlessotherwise noted. The deionized water (18.2 MO cm) was prepared using a water purification system (Milli-Q IQ7003; Merck
KGaA). UV-visible absorption spectra were recorded using a UVvisible absorption spectrophotometer (V-650; Jasco Corp. or UV1900i; Shimadzu Corp.). Fluorescence emission spectra wereobtained using a spectrofluorometer (FP-8300; Jasco Corp.).

The enzyme activity of Cat-HSA5 was evaluated by measuring
the H2O2 degradation based on the absorption intensity at
240 nm. The PBS solution (pH 7.4) of Cat-HSA5 (20 nM,
0.1 mL) was injected into H2O2 solution (5.3 mM, 1.9 mL,
PBS) in a quartz cuvette (10 mm optical path length). The
absorbance change at 240 nm was monitored for 2 min. The
same experiments were conducted at various H2O2 concentrations(0–50 mM). The initial rate constant (v0) was calculated using themolar absorption coefficient of H2O2 (e240 = 43.6 M 1 cm 1).35
From the relationship between [H2O2] 1 and v0 1 (Lineweaver–Burk plots), the catalytic activity (kcat/Km) was ascertained. The
control experiments were conducted using Cat (20 nM) under
the same conditions. Furthermore, the catalytic activity of
the Cat-HSA5 was evaluated by monitoring the concentrationof generated O2 using an O2-monitoring system (Oxygraph;Hansatech Instruments Ltd). First, the PBS solution of© 2022 The Author(s). Published by the Royal Society of Chemistry

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Cat-HSA5 (1 nM, 0.98 mL containing 0.001% Antifoam SI) was
poured into the electrode chamber. The dissolved O2 wasmeasured using gentle stirring under aerobic conditions at
25 1C. After confirming the O2 concentration level constant,
H2O2 in PBS (25 mM, 20 mL) was added to adjust the H2O2
concentration in the medium to 0.5 mM. Immediately after the
addition of H2O2, an increase in the dissolved O2 was observed.
The same experiments were conducted using various H2O2
concentrations (0–4 mM). The kcat/Km value of Cat-HSA5 wasdetermined from Lineweaver–Burk plots. The PBS solution of
Cat (1 nM) was also measured in the same manner.
Preparation of the Cat-(HSA-PP)5 cluster
The DMSO solution of PP (2 mM, 0.02 mL) was injected slowly
into the PBS solution (pH 7.4) of Cat-HSA5 (4 mM, 1.98 mL). The
obtained mixture was incubated for 3 hr with gentle stirring
under dark conditions at 30 1C, yielding Cat-(HSA-PP)5 (PP/CatHSA5 = 5.0, mol mol 1). The UV-vis absorption spectrum
revealed that PP is incorporated into the peripheral HSA
moieties of Cat-HSA5. HSA–PP was also synthesized in the samemanner.
Laser flash photolysis
Transient absorption spectra and their time-course measurements were recorded using a time-resolved spectrophotometer(TSP-1000; Unisoku Co. Ltd) with a Q-switched Nd: YAG laser(Minilite ML II; Continuum Inc.), which generates a secondharmonic (532 nm) pulse of 5–7 nm duration (10 Hz). The PBS
solution (pH 7.4) of Cat-(HSA-PP)5 ([PP] = 3 or 10 mM) was held
in a quartz cuvette (10 mm optical path length). For equilibration, N2 gas was flowed into the sample cuvette for 2 h. The
transient absorption spectra of the 3 mM solution were recorded
at 25 1C. Time courses at 452 nm after laser flash photolysis of
the 10 mM solution were measured at 25 1C. The triplet lifetime(tT) was determined using single exponential kinetics. The PBS
solution of HSA–PP was also measured similarly.
1O2 generation measurement

The formation of 1O2 was detected using ABDA. The DMSO
solution of ABDA was added to PBS solution (pH 7.4) of Cat(HSA-PP)5 ([PP] = 5 mM, [ABDA] = 50 mM, [DMSO] = 0.5 v/v%).
The samples were light irradiated using a LED lamp (KTL-350;
Kenko Tokina Corp.) equipped with an R-62 long pass filter(l 4 620 nm; Hoya Corp.) (25 mW cm 2 at 630 nm) with gentle
stirring using a magnetic stirrer. After 1, 5, 10, 30, and 60 min
irradiation, the UV-vis absorption spectra were recorded. From
the decrease of the absorbance at 380 nm, the residual ABDA
concentration was calculated. A sample without ABDA wasprepared simultaneously in all cases; its absorption change at
380 nm was subtracted from the data of the sample solution
with ABDA. The PBS solution of HSA–PP ([PP] = 5 mM, [ABDA] =50 mM) was measured similarly.

Materials Advances
Dulbecco’s Modified Eagle Medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), penicillin (100 unit per mL),
streptomycin (100 unit per mL), and amphotericin B(0.25 mg mL 1). The HeLa cells were seeded into a 96-well plate(seeding density; 7.5  103 cells per well in 100 mL) and wereincubated at 37 1C for 24 h. Then, after 100 mL of photosensitizer [Cat-(HSA5-PP)5 or HSA-PP in DMEM, [PP] = 0–50 mM] wasadded to each well, and the cells were incubated at 37 1C in thedark for 4 hr under air ([O2] = 20%) or hypoxic conditions ([O2] =1%) using a hypoxia culture kit (BIONIX; Sugiyama-Gen Co.,
Ltd). Subsequently, the cells were exposed to a LED lamp
equipped with an R-62 long pass filter (l 4 620 nm)(25 mW cm 2 at 630 nm) for 5 min, followed by 20 hr
incubation in the dark. After rinsing with PBS, 100 mL of the
CCK-8 reagent diluted ten-fold with DMEM was added to eachwell. Then the cells were incubated for 1 h. Finally, theabsorbance at 450 nm was measured using a microplate reader(Multiskan SkyHigh; Thermo Fisher Scientific Inc.).
The cell staining using trypan blue was conducted as follows. The supernatant in the well was carefully removed,
followed by adding 100 mL of 0.4 w/v% trypan blue solution.
After rinsing twice with 100 mL of PBS, optical microscopy
observations were performed. The same experiments were also
conducted without light irradiation.

Author contributions
T. K. designed and initiated this study. All the authors conducted the experiments and analysed the data. T. Y. and T. K.
drafted the manuscript.

Conflicts of interest
There are no conflicts to declare.

Acknowledgements
This work was supported by the Science Research Promotion
Fund from Promotion and Mutual Aid Corporation for Private
Schools of Japan, Grants-in-Aid for Scientific Research (B) (No.
21H01767) from JSPS, and a Joint Research Grant from theInstitute of Science and Engineering, Chuo University.

Notes and references

In a 5% CO2-95% air humidified incubator (MCO-5AC (UV);
Sanyo Electric Co., Ltd), HeLa cells were grown at 37 1C in the

1 D. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev. Cancer,
2003, 3, 380–387.
2 Q. Zhang, J. He, W. Yu, Y. Li, Z. Liu, B. Zhou and Y. Liu, RSC
Med. Chem., 2020, 11, 427–437.
3 X. Li, S. Lee and J. Yoon, Chem. Soc. Rev., 2018, 47,
1174–1188.
4 D. W. Zheng, B. Li, C. X. Li, J. X. Fan, Q. Lei, C. Li, Z. Xu and
X. Z. Zhang, ACS Nano, 2016, 10, 8715–8722.
5 L. Chen, S. F. Zhou, L. Su and J. Song, ACS Nano, 2019, 13,10887–10917.

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Paper

6 Y. Cheng, H. Cheng, C. Jiang, X. Qiu, K. Wang, W. Huan,
A. Yuan, J. Wu and Y. Hu, Nat. Commun., 2015, 6, 6–13.
7 Q. Chen, J. Chen, C. Liang, L. Feng, Z. Dong, X. Song,G. Song and Z. Liu, J. Controlled Release, 2017, 263, 79–89.
8 L. Liu, Y. Zhang, W. Qiu, L. Zhang, F. Gao, B. Li, L. Xu,
J. Fan, Z. Li and X. Zhang, Small, 2017, 13, 1701621.
9 A. Sahu, K. Min, J. Jeon, H. S. Yang and G. Tae, J. Controlled
Release, 2020, 326, 442–454.
10 Y. Matsumura and H. Maeda, Cancer Res., 1986, 46,
6387–6392.
11 H. Cabral, Y. Matsumoto, K. Mizuno, Q. Chen, M. Murakami,
M. Kimura, Y. Terada, M. R. Kano, K. Miyazono, M. Uesaka,
N. Nishiyama and K. Kataoka, Nat. Nanotechnol., 2011, 6,
815–823.
12 R. Zhang, R. Xing, T. Jiao, K. Ma, C. Chen, G. Ma and X. Yan,ACS Appl. Mater. Interfaces, 2016, 8, 13262–13269.
13 M. F. Attia, N. Anton, J. Wallyn, Z. Omran andT. F. Vandamme, J. Pharm. Pharmacol., 2019, 71, 1185–1198.
14 R. Haubner, W. A. Weber, A. J. Beer, E. Vabuliene, D. Reim,
M. Sarbia, K. F. Becker, M. Goebel, R. Hein, H. J. Wester,H. Kessler and M. Schwaiger, PLoS Med., 2005, 2, 0244–0252.
15 D. Tomita, T. Kimura, H. Hosaka, Y. Daijima, R. Haruki,
K. Ludwig, C. Böttcher and T. Komatsu, Biomacromolecules,
2013, 14, 1816–1825.
16 Y. Morita, T. Yamada, M. Kureishi, K. Kihira andT. Komatsu, J. Phys. Chem. B, 2018, 122, 12031–12039.
17 R. Funaki, T. Kashima, W. Okamoto, S. Sakata, Y. Morita,
M. Sakata and T. Komatsu, ACS Omega, 2019, 4, 3228–3233.
18 R. Haruki, T. Kimura, H. Iwasaki, K. Yamada, I. Kamiyama,
M. Kohno, K. Taguchi, S. Nagao, T. Maruyama, M. Otagiri
and T. Komatsu, Sci. Rep., 2015, 5, 1–9.
19 H. Iwasaki, K. Yokomaku, M. Kureishi, K. Igarashi,
R. Hashimoto, M. Kohno, M. Iwazaki, R. Haruki,
M. Akiyama, K. Asai, Y. Nakamura, R. Funaki, Y. Morita
and T. Komatsu, Artif. Cells, Nanomed., Biotechnol., 2018, 46,
S621–S629.

20 W. Okamoto, M. Hasegawa, T. Usui, T. Kashima, S. Sakata,
T. Hamano, H. Onozawa, R. Hashimoto, M. Iwazaki,
M. Kohno and T. Komatsu, J. Biomed. Mater. Res., 2022,
110, 1827–1838.
21 J. Ghuman, P. A. Zunszain, I. Petitpas, A. A. Bhattacharya,
M. Otagiri and S. Curry, J. Mol. Biol., 2005, 353, 38–52.
22 T. Yamada and T. Komatsu, ChemBioChem, 2021, 22,2526–2529.
23 E. Nagababu and J. M. Rifkind, Biochemistry, 2000, 39,
12503–12511.
24 W. A. Schroeder, J. R. Shelton, J. B. Shelton, B. Robberson,
G. Apell, R. S. Fang and J. Bonaventura, Arch. Biochem.
Biophys., 1982, 214, 397–421.
25 T. A. John, S. M. Vogel, C. Tiruppathi, A. B. Malik andR. D. Minshall, Am. J. Physiol., 2003, 284, 187–196.
26 A. Spada, J. Emami, J. A. Tuszynski and A. Lavasanifar, Mol.
Pharmacol., 2021, 18, 1862–1894.
27 M. Alfonso-Prieto, X. Biarnés, P. Vidossich and C. Rovira,J. Am. Chem. Soc., 2009, 131, 11751–11761.
28 A. Abuchowski, J. R. McCoy, N. C. Palczuk, T. van Es andF. F. Davis, J. Biol. Chem., 1977, 252, 3582–3586.
29 P. A. Zunszain, J. Ghuman, T. Komatsu, E. Tsuchida andS. Curry, BMC Struct. Biol., 2003, 3, 1–9.
30 J. Hu, R. Allen, S. Rozinek and L. Brancaleon, Photochem.
Photobiol. Sci., 2017, 16, 694–710.
31 J.-H. Fuhrhop, C. Demoulin, C. Böttcher, J. Köning and
U. Siggel, J. Am. Chem. Soc., 1992, 114, 4159–4165.
32 N. A. Kuznetsova, N. S. Gretsova, O. A. Yuzhakova,
V. M. Negrimovskii, O. L. Kaliya and E. Luk’yanets, Russ.
J. Gen. Chem., 2001, 71, 36–41.
33 S. Wang, F. Yuan, K. Chen, G. Chen, K. Tu, H. Wang and
L. Q. Wang, Biomacromolecules, 2015, 16, 2693–2700.
34 S. Z. F. Phua, G. Yang, W. Q. Lim, A. Verma, H. Chen,
T. Thanabalu and Y. Zhao, ACS Nano, 2019, 13, 4742–4751.
35 I. Hara, N. Ichise, K. Kojima, H. Kondo, S. Ohgiya,
H. Matsuyama and I. Yumoto, Biochemistry, 2007, 46, 11–22.

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