Cascade Reactions by Nitric Oxide and Hydrogen Radical for Anti-Hypoxia Photodynamic Therapy Using an Activatable Photosensitizer

Jian Sun, Xuetong Cai, Chengjun Wang, Ke Du, Weijian Chen, Fude Feng,* and Shu Wang*

Article

Cascade Reactions by Nitric Oxide and Hydrogen Radical for Anti-

Hypoxia Photodynamic Therapy Using an Activatable

Photosensitizer

Cite This: J. Am. Chem. Soc. 2021, 143, 868 −878

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sı Supporting Information

ABSTRACT: Organelle-targeted activatable photosensitizers are attractive to

improve the speciﬁcity and controllability of photodynamic therapy (PDT),

however, they suﬀer from a big problem in the photoactivity under both

normoxia and hypoxia due to the limited diversity of phototoxic species

(

mainly reactive oxygen species). Herein, by eﬀectively photocaging a π-

conjugated donor−acceptor (D−A) structure with an N-nitrosamine

substituent, we established a unimolecular glutathione and light coactivatable

photosensitizer, which achieved its high performance PDT eﬀect by targeting

mitochondria through both type I and type II (dual type) reactions as well as

secondary radicals-participating reactions. Of peculiar interest, hydrogen

radical (H ) was detected by electron spin resonance technique. The

generation pathway of H via reduction of proton and its role in type I

•

reaction were discussed. We demonstrated that the synergistic eﬀect of

multiple reactive species originated from tandem cascade reactions comprising reduction of O by H to form O ^•⁻/HO ^•and

•

−

•

−

downstream reaction of O₂with NO to yield ONOO . With a relatively large two-photon absorption cross section for

photoexcitation in the near-infrared region (166 ± 22 GM at 800 nm) and ﬂuorogenic property, the new photosensitizing system is

very promising for broad biomedical applications, particularly low-light dose PDT, in both normoxic and hypoxic environments.

INTRODUCTION

against hypoxic tumor cells, attributed to the limitation of the

■

type II photosensitizing pathway that needs an aerobic

Photodynamic therapy (PDT) is a therapeutic technique that

applies a light-sensitive drug (photosensitizer, PS) and a light

source to achieve selective destruction of abnormal cells,

wherein the exposure of PS to light in the presence of tissue

oxygen generates reactive oxygen species (ROS) and other

cytotoxic free radicals. PDT has received more attention due to

its unique features, including high biocompatibility, high

spatiotemporal selectivity, and minimal invasiveness.

There are a variety of PSs that are clinically approved and

have been successfully applied (e.g., Photofrin, acridine orange,

environment to produce singlet oxygen ( O ). Simultaneously,

the additional advantages that can be achieved with targeting

of PSs or context-speciﬁc activation that thereby provide a dual

6,7

selective eﬀect.

The obstacle for oxygen-dependent PSs can be overcome by

a type I photosensitizing mechanism that favors the ﬁghting of

hypoxia to improve PDT through the generation of toxic free

−3

•

radicals such as hydroxyl radical ( OH), superoxide ion

•−

•

(

O₂), and hydroperoxyl radical (HO ). In view of the

reported examples collectively enumerated in Table S1 of the

Supporting Information (SI ), most of the type I reaction-based

PSs are precious metallic complexes and metal-centered

-ALA, and mTHPC) for treating cancers such as skin cancers,

prostate cancers, and head and neck cancers. PSs are critical

to the eﬀect of PDT in tumor ablation using light to induce

transient photochemical processes. Most ROS, an umbrella

term of oxygen-derived short-lived intermediates reactive to

biocomponents, are biorelevant in redox signaling, biosyn-

thesis, and metabolic homeostasis and are enzymatically

controlled at a dynamic and ﬁnely regulated level in living

macrocycles, on the basis of the redox photochemistry that

have been intelligently evolved by nature using light harvesting

capability of chlorophyll to initiate photosynthesis. In

contrast, the development of nonmetallic PSs that relieve the

Received: October 2, 2020

Published: January 8, 2021

cells. In the PDT eﬀect, the photogenerated ROS in the

spatially targeted area of light exposure exceeds the scavenging

ability of enzymes and endogenous reductants; causes

irreversible damage to proteins, lipids, and nucleic acids; and

ultimately results in cell death. However, traditional PSs face a

major obstacle in that they have poor therapeutic eﬃcacy

2021 American Chemical Society

J. Am. Chem. Soc. 2021, 143, 868−878

Figure 1. (a) Chemical structure of DANO and schematic illustration of GSH−light coactivation for enhanced PDT. (b) Synthetic route of DAPS

and DANO. (c) Transformation of 3b from 3 to verify the site of N-alkylation.

concerns with metal toxicity remains in its infancy in

compared to normal cells (millimolar versus submillimolar

concentrations), GSH-dependent photoactivation potentially

reduces the risk of nonspeciﬁc phototoxicity.

10−13

counteracting hypoxia.

Particularly, for type I reactions

producing O₂^•, it is necessary to maximize the O₂mediated

−

•−

toxicity via one or more secondary reactions that aﬀord

−

strongly reactive species such as peroxynitrite (ONOO ) and

RESULTS AND DISCUSSION

•

■

OH, in addition to its reactivity to transition metals (e.g.,

reduction of iron complexes in cytochrome c and oxidation of

iron−sulfur clusters). To improve the responsive selectivity,

the development of new activatable photosensitizers (aPSs)

responsive to the endogenous and/or exogenous stimuli (e.g.,

pH, redox environment, and enzymes) is one of the most

Synthesis and Characterization of DANO. A compact

D−A structure with maximum absorption at the blue light

region is designed for this study as D−A structures have

suﬃcient sites for ﬂexible functionalization and feature intense

intramolecular charge transfer (ICT) absorption for blue

light excitation as well as a TPE that is compatible with an 800

nm laser. Using benzodithiophene (BDT) and benzotriazole

,15

attractive strategies.

Currently, aPS molecules that enable

generation of diverse reactive species, not limited to ROS, are

urgently needed for eﬃcient PDT under hypoxia. We recently

reported a glutathione-hydrogen peroxide (GSH−H O )

(

BTz) as the electron donor and acceptor, respectively, we

designed a conjugated BDT−BTz structure with an −N(Me)−

NO substituent positioned at the BTz ring such that the whole

molecule could be in a stable and deactivated state. The TPP

cations are attached to the BDT moiety for localized

accumulation into mitochondria which are known as one of

coresponsive benzothiadiazole-based aPS that could be

activated to its mature form (derived from HO−NH ArSO H

structurally shown in Table S1 ) with dual type photosensitiz-

ing capability. However, endogenous H O is not suﬃcient

best targets for PDT.

enough for aPS activation.

It is noteworthy that mitochondria are less enriched with

molecular oxygen as compared to the cytosol owing to the

considerable oxygen consumption by the respiratory chain,

In the present work, we reported an organic functional

molecule, termed DANO, that possesses a π-conjugated

donor−acceptor (D−A) backbone equipped with an N-

nitrosamine substituent and two amphiphilic triphenylphos-

phine (TPP) ligands (Figure 1a). DANO has following

important features: (1) the D−A backbone is photocaged by

the N-nitroamine substituent, and photouncagable to liberate

gaseous NO in a GSH dependent manner; (2) the activated

DANO forms a dual type PS to eﬀectively generate ROS (e.g.,

O and O ) as well as reactive nitrogen species (RNS) such

as NO and peroxynitrite (ONOO ); (3) upon activation of

which necessitates type I or dual type reaction for oxygen-

•−

independent PDT. With a pK of 4.8, O₂is negatively

charged at neutral pH, with the membrane impermeable and

mostly conﬁned in the mitochondrial compartment. As

mitochondrial superoxide mutase (Mn-SOD) exists at high

•

•−

concentrations (5−20 μM) and scavenges O₂with an

extremely high second-order reaction constant (k = 1.2 × 10

•−

−1 −1

•−

•

s ), mitochondrial O₂is short-lived. However, for the

−

case of DANO, this scavenging issue can be relieved for a

•

•−

•

−

DANO by light and GSH, hydrogen radical (H ), assigned to

couple of reasons: (1) O reacts with NO to form ONOO

−1 −1

reactive hydrogen species (RHS), is another detectable reactive

intermediate, which is an extremely rare ﬁnding in PDT and

adds more insight and understanding into the origination of

ROS involved in type I reaction and the mechanism of

multicomponent participating synergistic eﬀects for PDT. The

cascade reactions and combination of the above collective

features with outstanding properties of near-infrared (NIR)

two photon excitation (TPE) and imaging make DANO

promising for enhanced PDT in complex tumor environments.

Given that GSH is often at elevated levels in tumor cells as

(k = 6.7 × 10 M s ), favored by the simultaneously

•

−

•

colocalized generation of O and NO. The slow oxidation

•

of NO by O can not compete with this reaction. (2)

ONOO inactivates Mn-SOD through nitration reaction,

which potentially suppresses SOD-catalyzed scavenging of

−

•

−

O₂. Both O and ONOO are strong reactive oxygen/

nitrogen species (RONS), but travel at diﬀerent distance limits

and target diﬀerent biomacromolecules associated with

mitochondrial functioning. Usually, O causes lipid oxidation

and selective photodamage to certain amino residues of

J. Am. Chem. Soc. 2021, 143, 868−878

Figure 2. (a) UV−vis absorption (solid line) and ﬂuorescence spectra (dashed line) of DANO (orange line) and DAPS (blue line) in 10%

acetonitrile−PBS (pH 7.4). (b) Fluorescence enhancement (I/I ) of the mixture containing DANO (5 μM) and GSH (0−500 μM) upon LED

light irradiation at 20-s intervals. (c) UV−vis spectra and (d) UPLC analysis of solutions containing DANO (5 μM) and GSH (200 μM) upon

LED light irradiation. (e) Changes of ﬂuorescence intensity of DAF-2 solutions containing DANO (5 μM) and GSH (0 or 200 μM) with or

without LED light irradiation. (f) ESR signal of the mixture containing DANO (100 μM) and PTIO (500 μM) under LED light irradiation. The

−

reaction solvent was 10% acetonitrile−PBS (pH 7.4). Light irradiation proceeded under a 405 nm LED with a power density of 20 mW cm .

−

proteins, while ONOO induces protein nitration at tyrosine

residues and oxidatively inactivates mitochondrial proteins

(

nonﬂuorescent (Φ < 0.001 in methanol, using coumarin

307 with Φ = 0.56 in ethanol as a standard) owing to the

25,26

e.g., electron transport chain components).

N-Nitrosoaminophenol and nitroaniline derivatives have

been developed as NO photoreleasers.

those NO releaser−linker−reporter dyads, the −N(Me)−NO

functionality of DANO has distinctive features and roles: (i)

photocaging BTD−BTz backbone through photoinduced

electron transfer (PET); (ii) responsive to the mutual presence

of light irradiation and GSH rather than the light irradiation

caging eﬀect of the N-nitrosamine group. DAPS absorbed

−1

maximally at 392 nm (ε₃₉₂= 3.9 × 10 M cm ), with

comparable intensity at 405 nm to DANO, which means both

DANO and DAPS were compatible with a 405 nm light

emitting diode (LED) for photochemical reactions. Diﬀerent

•

27−30

As compared to

•

from DANO, DAPS was ﬂuorogenic (Φ = 0.047 in PBS) and

emissive maximally at 532 nm, bright enough for cell imaging

purposes.

•

alone; and (iii) cascading NO release and generation of

RONS (e.g., O , O , and ONOO ) and RHS (i.e., H ). The

With GSH and light as stimuli factors, the uncaging process

of DANO over a period of 120 s was investigated in 10%

acetonitrile-phosphate saline buﬀer (PBS, pH 7.4). Under light

•−

−

•

important role of GSH in the regulation of NO release and

the photochemical process are taken into account, which

makes DANO exceptional for optimal PDT.

The synthesis of DAPS and DANO was shown in Figure 1b.

The acceptor segment 3 was prepared in three steps (overall

yield 15% from 1), and coupled to the donor segment 4 via

Pd(0)−catalyzed Stille reaction to produce conjugated

structure 5. After substitution reaction (−Br → −TPP),

DAPS was obtained. N-Nitrosylation reaction was carried out

by treating DAPS with sodium nitrite in acetic acid to aﬀord

DANO in a yield of 85%. It should be noted that N-alkylation

of asymmetric benzotriazole could occur at the three nitrogen

positions, which raised a concern about possible impurities

−

(λ 405 nm, 20 mW cm ), the increase of GSH concentration

(0 −0.5 mM) promoted ﬂuorescence turn-on response (Figure

S2 ), causing ﬂuorescence enhancement up to 30-fold (Figure

2b). The reaction kinetics was also analyzed by UV−vis

absorption spectra (Figure 2c). At a ﬁxed GSH concentration

(0.2 mM), the ICT absorption band gradually blue-shifted

with enhanced intensity. Interestingly, an isosbestic point

appeared at 370 nm, well matching the intersection of the two

absorption curves of DANO and DAPS (Figure 2a). The

DANO → DAPS conversion was conﬁrmed by ultrahigh

performance liquid chromatography−mass (UPLC−MS)

analysis (Figure 2d) through quantitatively monitoring the

consumption of DANO and concomitant production of DAPS

(

i.e., isomerized forms) of key intermediate 3. To address this

issue, we treated 3 by bromination and deamination reactions

at the retention time (t ) of 2.90 and 2.84 min, respectively.

and achieved the dibromo-substituted intermediate 3b (Figure

Evidently, the reaction was fast and rather clean, complete after

c) which was identiﬁed by the NMR analysis (Figure S1),

120 s of light irradiation. The only minor product was detected

thereby conﬁrming complete N-alkylation at the N2 position.

Photoactivation of DANO. DANO exhibited an intense

absorption band (Figure 2a) assigned to the ICT transition,

at t 2.30 min, and identiﬁed to be a GSH adduct (DASG) by

the evidence of high resolution mass spectrum (HRMS, m/z

710.7332, Figure S3). However, in the absence of GSH, the

−1

peaked at 405 nm (ε₄₀₅= 3.1 × 10 M cm ), and nearly

conversion rate was only 12% at an irradiation time (t ) of 120

irr

J. Am. Chem. Soc. 2021, 143, 868−878

Journal of the American Chemical Society

Figure 3 . Only L-cysteine led to similar ﬂuoroescence

Article

s, without a detectable signal at t 2.30 min (Figure S4) as GSH

was essential for the formation of the GSH adduct. In the dark,

no product was detected (Figure S5).

generation, the carbon-centered radical was also detected by

the six-line signal (a = 15.4 mT, a = 22.2 mT).

N βH

We speculated that the formation of the carbon-centered

radical was associated with the aniline radical resulting from

the homolysis of the N−NO bond upon exposure of DANO to

light and played a key role in production of DASG (i.e., the

GSH adduct, detectable by UPLC−MS). Therefore, the overall

photoreaction process was proposed in Figure 4b. In brief,

GSH acts as a scavenger of DA1^•to move forward the

reversible N−NO homolysis reaction and produce the major

product DAPS. The reversibility of the N−NO homolysis

reaction was evidenced by the inhibition assay using NOC-13

as a NO donor (Figure S6 ). In another pathway, the carbon-

The above results demonstrated that GSH and light could

co-activate DANO by brief light irradiation within 120 s, with

negligible degradation of DAPS (Figure 2d) in an aerobic

•

photoactivation condition. To characterize the NO photo-

release property of DANO, we carried out the following

•

experiments. First, DAF-2, an NO speciﬁc probe, was

observed to ﬂuorescently respond to the irradiation cycles

applied to DANO, particularly in the presence of GSH (Figure

e). The ﬂuorescence intensity was plateaued after an overall

•

t_i_r_rof 100 s to indicate complete NO release, in accord with

•

the UPLC data (Figure 2d). Next, electron spin resonance

centered radical DA2 resonated with DA1 is scavenged by

•

(

ESR) spectra were measured, using 2-phenyl-4,4,5,5,-

GS to form DASG in a minor quantity.

tetramethylimidazoline-1-oxyl 3-oxide (PTIO radical) as a

spin trapper, which indicated NO-mediated generation of

One surprising ﬁnding is the concurrent photogeneration of

H , which likely involves a proton reduction process associated

•

PTI radical after photoreaction (t = 1 or 2 min) (Figure 2f).

with the charge separation property of D−A type materi-

irr

39,40

The selectivity of DANO for GSH in ﬂuorescence response

after light irradiation over a broad range of biologocally

relevant species was investigated, and the results were shown in

als.

The electron transfer could occur from the environ-

ment (e.g., high concentration of GSH) to the long-lived

triplet excited state of DAPS (termed DAPS*, originating

from the singlet excited state, termed DAPS*, via intersystem

•

−

crossing process) and generate DAPS , followed by trans-

ferring one electron to proton and ﬁnally returning to the

ground state of DAPS (Figure 4c). Generally, as the smallest

free radical and strong reductant, the short-lived H^•is

immediately converted to secondary radicals or products. As

•

−1

DMPO captured H at a rather high rate (k = 3.8 × 10 M

−

s ), the ESR measurement could be a useful tool to identify

H . Another evidence of H generation was provided by the

HRMS data (Figure S7) that indicated formation of the

•

TEMPO adduct (m/z 158.1545) of H . To provide further

experimental evidence, we investigate the hydrogenation

capability of hydrogen radical toward the aryl carbon−carbon

double bond, using 4-vinylbenzoic acid (PVBA) as a substrate.

As shown in Figure S8 , after photoreaction of the mixture

containing DANO, GSH, and PVBA, hydrogenated PVBA is

detected by HR−MS with an m/z of 149.0594, in excellent

consistency with the predicted value (m/z 149.0608), but it is

not detectable in the control experiment lacking light

irradiation. This result conﬁrms the presence of the photo-

generated hydrogen radical.

Figure 3. Fluorescence enhancement (I/I ) of the mixture solutions

containing DANO (5 μM) and diﬀerent biologically relevant species

(

200 μM) upon LED light irradiation (1 min, λ 405 nm, 20 mW

−

cm ). 1: Control (DANO only in dark); 2: DANO only in light; 3:

NO ; 4: NO ; 5: SO ; 6: Fe ; 7: Fe ; 8: Zn ; 9: Cu ; 10:

H O ; 11: ClO ; 12: Gly; 13:Glu; 14: Arg; 15: NADH; 16: Cys; and

−

2−

−

17: GSH.

Despite the fact that H^•is one of the intermediates

42−44

generated by relatively harsh protocols

such as ionizing

irradiation, atmospheric plasma treatment, photolysis of water

by high energy UV irradiation, and photocatalysis, H is hardly

enhancement to GSH. Taking into account that GSH and L-

cysteine are the most abundant intracellular thiols and the

typical intracellular level of GSH (1−10 mM) is almost 1 or 2

orders of magnitude higher than that of L-cysteine (lower than

•

available in aqueous solutions under mild conditions.

Presently, lacking controllable in situ generation methods,

the reactivity of H^•with cellular components is largely

5−37

00 μM),

GSH is considered to be the most responsive

•

unkown. In living systems, H participating reactions, such as

thiol species to activate DANO in the living cells.

−1 −1 45

•

extremely fast reduction of O (k = 2.1 × 10 M s ) into

H Generation and Theoretical Calculation. To look

•

•−

^H^O2 (i.e., protonated O ) should be of biological value.

Highly lipophilic HO is detrimental to lipids by perox-

idation. ^I^tⁱ^sⁿ^o^t^e^d^t^h^a^t^O2 ^/^H^O2 was not detectable when

H was depleted by excess DMPO (Figure 4a), implying that

more closely into the mechanism of photochemistry and roles

of GSH in the photoactivation of DANO, we performed ESR

spin trapping with a high concentration of 5,5-dimethyl-1-

pyrroline N-oxide (DMPO, 500 mM) that can convert free

radical intermediates into long-lived spin adducts. Interest-

•

•−

•

−

direct electron transfer between DAPS and O to produce

•

−

•

^O2 ^/^H^O2 in the presence of excess GSH is unfavorable but

ingly, under light (t = 1 min), the ESR signal was nearly silent

irr

•

mediated by H as follows:

in the absence of GSH, but emerged as a combination of two_•

groups of peaks if GSH was present (Figure 4a). DMPO−H

adduct was identiﬁed according to the characteristic hyperﬁne

•

•−

H + O → HO ↔ H + O

(1)

splitting constants of the nine-line signal (a = 16.2 mT, a

To understand the photocaging mechanism of the −NO

functionality, we carried out theoretical calculations based on

time-dependent density functional theory (TD-DFT) at the

βH

•

2.1 mT),

which suggested the emergence of H .

•

Interestingly, as the ESR data revealed, accompanied by H

J. Am. Chem. Soc. 2021, 143, 868−878

Figure 4. (a) ESR signal of the mixture containing DANO (1 mM), GSH (0 or 1 mM), and DMPO (500 mM) in 40% acetonitrile−PBS under

−

LED light irradiation (λ 405 nm, 20 mW cm , t = 1 min). (b) Plausible mechanism of DANO → DAPS/DASG transformation mediated by

irr

•

GSH and light. (c) Proposed mechanism for the GSH-dependent photogeneration of H . (d) Schematic diagram for frontier molecular orbitals and

electronic transitions of DAPS on a basis of TD−DFT calculation.

−

level of B3LYP/6-31G (d) (Table S2). For DAPS, as shown in

Figure 4d, the intersystem crossing (ISC) process is favorable

the same light irradiation protocol (λ 435 nm, 1.5 mW cm ).

The generation of O₂^•and O was also conﬁrmed by ESR

−

by the small singlet−triplet energy gap (ΔE , 0.09 eV)

analysis (Figure 5c,d), using DMPO (500 mM) and 2,2,6,6-

tetramethyl-4-piperidone (TEMP) as spin trapping agents,

respectively. In contrast, the ESR signal was silent for DANO

between S and T excited states, allowing the type I/type II

pathway to take place. The S → S transition (corresponding

in both O₂^•and O detection. ROS such as OH were not

−

•

to H → L transition) is allowed with a relatively high oscillator

strength (f = 0.5243), which predicts that DAPS is a

ﬂuorogenic PS. In both HOMO and LUMO orbitals, electron

delocalization over the π-conjugated D−A backbone and the

nitrogen atom of the −NH(Me) substituent favors photo-

induced charge transfer. However, DANO is another case,

associated with the addition of −NO to the −NH(Me)

substituent. First, in comparison to DAPS, the dihedral angle

between BDT and BTz planes is enlarged from 15.0° to 27.5°.

Second, photoexcitation causes drastic electron transfer from

the BDT core to the BTz plane and an N−NO bond. In the

LUMO orbital, electrons are localized into the acceptor

segment (Figure S9). Additionally, the H → L transition is

nearly forbidden (f = 0.0008). Consequently, the N−NO bond

brings electronic and structural eﬀects by reversing the electron

donating eﬀect of the −NH(Me) substituent, facilitating the

PET process, causing geometrical changes of the D−A

skeleton and caging the optical properties of the conjugated

structure.

detected by ESR measurement (Figure S11). These results

•

−

indicated that DAPS is an eﬃcient dual type PS, with O and

O as the major ROS. Comparing the DANO/GSH reaction

system (Figure 4a) and DAPS alone in the presence of the

_s_a_m_e_c_o_n_c_e_n_t_r_a_t_i_o_n_o_f_D_M_P_O₍₅₀₀_m_M₎_,_H• and O₂•−

adducts were detected, respectively, which was attributable to

the role of excess GSH. Given the high concentration of

endogenous GSH (1−10 mM) in living cells, we inﬀered that a

•

cascade reaction of GSH-dependent H and molecular oxygen

accounts for the generation of O₂/HO₂^•(eq 1).

•−

ESR measurement was not applicable to the detection of

−

ONOO which was a nonradical substance. Alternatively,

−

photogenerated ONOO in the DANO solution over a period

−

of 120 s under light irradiation (λ 405 nm, 10 mW cm ) was

−

detected by TPNIR-FP, an ONOO speciﬁc probe reported by

Tang et al., according to the ﬂuorescence ﬂuctuation at 635

nm (Figure S12). The ﬂuorescence response was positively

dependent on the GSH concentration (Figure 5e). However,

the eﬀects of excess GSH were double edged. On the one

hand, GSH promoted the photoactivation of DANO to release

Detection of RONS. Upon light irradiation, RONS

including O₂^•

−

O , and ONOO were detected by

−

ﬂuorescent probes or ESR-based methods. Using dihydroethi-

•

−

NO and restore DAPS, thereby substantially accelerating the

dium (DHE) as a ﬂuorescent probe for O₂, DAPS was

−

formation of ONOO . On the other hand, GSH competed

shown to have stronger type I photosensitizing capability

−

(

Figure 5a) than methylene blue and HO−NH ArSO H (a

with TPNIR-FP in the reaction with ONOO (k = 5.8 × 10

−1 −1

dual type PS). Meanwhile, DAPS exhibited O photosensitiz-

ing capability (Figure 5b) with a O generation eﬃciency

s ), which led to an underestimation of the level of

−

generated ONOO .

(

Φ ) of 0.42 in methanol (Figure S10), using 1,3-

To conclude, the photouncaging of DANO is coactivatable

diphenylisobenzofuran (DPBF) as a O trapper and Ru-

by light irradiation and GSH, and DAPS is a PS eﬀective in a

•

(

bpy) Cl (Φ 0.87 in methanol) as an actinometer under

dual type mechanism. The interplay of H and O opens a

J. Am. Chem. Soc. 2021, 143, 868−878

photogeneration of H , O /HO , NO, ONOO , and O by coactivation of DANO with GSH and light.

Article

−

Figure 5. (a) The plot of ﬂuorescence ﬂuctuation (ΔF) of DHE at 620 nm versus t . The LED light power density was 10 mW cm , and the

irr

irradiation wavelengths were 405, 405, and 635 nm for DAPS, HO−NH ArSO H, and methylene blue, respectively. The concentration of each

photosensitizer was 5 μM. (b) The plot of A/A of DPBF versus t , where A and A denoted absorption intensity at 410 nm before and after light

irradiation, respectively. LED light irradiation (λ 435 nm, 1.5 mW cm ) was used. ESR spectra for the detection of (c) O and (d) O using

irr

−

•−

DMPO (in DMSO) and TEMP (in H O) as spin trapping probes, respectively. (e) Fluorescence enhancement of the mixture containing TPNIR-

−

−2

FP (ONOO probe) and indicated substrates after LED light irradiation (λ 405 nm, 20 mW cm , t = 0−120 s). (f) Schematic illustration of the

irr

•

•−

•

−

−

Figure 6. (a) CLSM images of DANO-treated HeLa cells after LED light irradiation (λ 405 nm, 20 mW cm , t = 0, 1 and min). The cells were

irr

stained with Mito Red Tracker. Scale bars, 10 μm. (b) The median ﬂuorescence intensities derived from images in a. Statistical signiﬁcance was

assessed using a two-way analysis of variance test (**P ≤ 0.05, ***P ≤ 0.001). (c) Fluorescence images of intracellular RONS. Scale bars, 50 μm.

door for a cascade of NO and O₂^•⁻to generate ONOO . The

schematic diagram of networked ROS/RNS/RHS generation

pathways was concisely illustrated in Figure 5f.

•

−

Intracellular Activation of DANO and Phototoxicity.

The activation process was investigated by imaging the living

cells with confocal laser scanning microscopy (CLSM). HeLa

cells were treated with DANO (5 μM) for 2 h, washed, and

J. Am. Chem. Soc. 2021, 143, 868−878

Figure 7. (a) CLSM images of HeLa cells treated with DANO (0 or 5 μM) after LED light irradiation (t_i_r_r= 0 or 1 min) or treated with CCCP.

The cells were stained with JC-1. Scale bars: 10 μm. (b) Viabilities of HeLa cells treated with varied concentrations of DANO (0−5 μM) 24 h after

LED light irradiation (t = 0 or 4 min). (c) Viabilities of HeLa cells treated with DANO (5 μM) under 21% or 1% O condition, 24 h after LED

irr

light irradiation (t_i_r_r= 0−5 min). (d) Fluorescence imaging of DANO (5 μM)-treated HeLa cells after LED light irradiation (t_i_r_r= 0−5 min). The

live and dead cells were stained by Calcein-AM and PI, respectively. Scale bars, 100 μm.

−

buﬀer), and TPNIR-FP as ﬂuorescent probes for NO, O₂^•⁻,

•

received brief light irradiation (λ 405 nm, 1.5 mW cm ). The

mitochondria were stained with Mito Tracker Red. DAPS

could be detected by its green ﬂuorescence collected at 470−

−

O , and ONOO , respectively. As a result, DANO-treated

cells in the dark showed similar ﬂuorescence intensity to the

background signal (Figure 6c), however, responding to light

irradiation (t_i_r_r= 1 min) with enhanced ﬂuorescence that

indicated the respective existence of photogenerated RONS.

The level of total intracellular RONS was reﬂected by the

elevated ﬂuorescence signal using 2,7-dichloroﬂuorescein

diacetate (DCFH-DA) as a nonspeciﬁc probe. These results

revealed that the photoactivation of DANO in the living cells

was successful and multiple toxic RONS were generated,

thanks to the eﬀective type I and type II reactions secondary to

40 nm ranges with excitation at 405 nm. As compared to the

dim ﬂuorescence of the control group (t_i_r_r= 0 min), the

irradiated cells (t = 1 min) emitted bright green ﬂuorescence

with a Pearson’s overlap coeﬃcient (R ) of 0.82 (Figure 6a,b),

irr

which indicated successful activation of DANO in mitochon-

−

dria by a low dose of light (1.2 J cm ). Using Mito Tracker

Red as a ﬂuorescent indicator, it seems the mitochondrial

membrane of DANO-treated cells was destabilized after 1−2

min of light irradiation. The GSH-responsive property of

DANO was investigated using L-buthionine-(S,R)-sulfoximine

BSO) as a GSH depletor. As shown in Figure S13, treatment

by BSO (10 μM) led to a decrease of ﬂuorescence intensity in

•

•−

photorelease of NO. Particularly, the detected signals of O

−

•

(

and ONOO conveyed a message that the H and NO

cascade reactions could occur in the living system, which

provides a foundation for synergistic antihypoxia PDT.

Depolarization of the mitochondrial membrane is one of

leading pathways for mitochondria-localized PS to induce cell

apoptosis. To evaluate the eﬀect of photoexcited DANO on

mitochondria, we examined the changes of the mitochondrial

the DANO-treated cells upon light irradiation (t 1 min, λ 405

nm, 20 mW cm ) by ∼25%, in agreement with the GSH-

irr

−2

dependent activation manner of DANO.

Note that a relatively longer irradiation time (t_i_r_r= 2 min)

would decrease the ﬂuorescence intensity but increase the

cytotoxicity of DANO. This observation could be interpreted

by the eﬀect of mitochondria-targeting PDT, according to the

morphological changes of mitochondria from a tubular state

favorable for staining) to a fragmented state (not favorable for

staining) (Figure 6a), which was well consistent with the

previous report of PDT by modulation of the mitochondrial

membrane potential (MMP, ψ ) with commercial dye JC-1 as

a Δψ indicator by comparing the emission signals of J-

monomer (in the green channel, predominantly distributed in

cytosol) and J-aggregates (in the red channel, located in intact

mitochondrial membrane) under CLSM. In the dark, bright

red ﬂuorescence was detected in DANO-treated cells, in a

similar pattern to the control experiment (Figure 7a). The

intensity of red ﬂuorescence was sharply attenuated in the

DANO-treated cells exposed to blue LED light (λ 405 nm, 20

(

morphology. The light irradiation activated DANO and

caused liberation of highly cytotoxic species that led to leakage

of ﬂuorescent molecules and aﬀected the integrity of

mitochondrial membrane structures and also probably the

−

mW cm , t = 1 min).

irr

cell membrane, as previously revealed by Zhao et al. As a

result, the ﬂuorescence channels from both the Mitotracker

and the ﬂuorescent product by activating DANO showed

The photodamage eﬀect was comparable to the eﬀect of

CCCP (a mitochondria membrane disrupter). The prompt

changes of ψ were attributable to the permeabilization of the

reduced intensity (t = 2 min).

To assess the intracellular RONS level, we detected primary

RONS using DAF-FM DA, DHE, singlet oxygen sensor green

mitochondrial membrane, induced by the eﬀect of locally

generated ROS on the mitochondrial components such as the

permeability transition pore complex (PTPC) including a

voltage-dependent anion channel (VDAC, the most abundant

irr

(SOSG, cell permeable in standard maintenance medium

J. Am. Chem. Soc. 2021, 143, 868−878

Journal of the American Chemical Society

mitochondrial outer membrane protein) and adenine nucleo-

tide translocator (ANT, the most abundant mitochondrial

inner membrane protein). For example, ANT is extremely

sensitive to O -mediated oxidation at the thiol groups

responsive for nucleotide transport and opening of the

permeability transition pore. Peroxidation of unsaturated

•

−

lipids by excess RONS (e.g., O , HO and ONOO ) can

also induce mitochondrial dysfunction and ψ disruption. The

combined eﬀect of RONS tends to impair a broad spectrum of

critical mitochondrial targets.

To investigate the in vitro PDT performance of DANO, we

carried out MTT assay 24 h after treatment of HeLa cells with

DANO over various periods of light irradiation (λ 405 nm, 20

−

mW cm for 0−5 min). The dark toxicity of DANO (0−5

μM) was low (Figure 7b). Light irradiation (t = 4 min) of

irr

DANO- treated cells caused decreased viabilities, strickingly

reducing the viability to only 6% in the presence of 5 μM

DANO. The phototoxicity was also dependent on the light

−

ﬂuence (0−6 J cm ) and oxygen concentration (Figures 7c

and S14 ). Although the PDT eﬀect was more favored under

1% O than 1% O because of O generation, the cell killing

−

performance under 1% O (only 9% cell survival; 6 J cm )

was comparable to or prevailing over the most reported PDT

agents shown in Table S1. The PDT eﬀect under 21% O and

% O was also veri ﬁed by the live/dead cell staining assay

(Figures 6d and S15 ) which recognized dead cells (in red)

from live cells (in green), highlighting the excellent PDT

performance of activated DANO under both normoxia and

hypoxia. DAPS exhibited signi ﬁ cant phototoxicity under both

normoxia and hypoxia (Figure S16), thanks to its photo-

sensitizing capability through the dual type pathway. However,

the PDT eﬀect was lower than DANO.

In Vitro Two-Photon Imaging and PDT. Two-photon

property is attractive for aPSs by excitation in an NIR window

and localized region with improved penetration depth. With a

D−A conjugated backbone contributable to two-photon

blue light to activate multiple photochemical reactions

secondary to NO release; (3) the dually eﬀective photo-

•

reaction pathways (type I and type II) enable photogeneration

•−

•

−

of reactive O/N/H species such as O , O /HO , ONOO ,

absorption, DAPS exhibited a large δ value of 166 ± 22

•

and H ; (4) DANO targets mitochondria and exhibits

excellent in vitro PDT performance under both normoxia

GM at 800 nm (in methanol, using rhodamine 6G as a

reference ). To make sure of the two-photon photoactivation

with DANO, we loaded DANO solution containing 0 or 200

(

21% O ) and hypoxia (1% O ) by inducing apoptosis of

tumor cells (<10% cell viability) at a very low dose of light up

to 6 J cm ; and (5) eﬃcient two-photon excited PDT with

μM GSH into a glass capillary (0.3 mm in diameter) for

−2

irradiation by an 800 nm two-photon laser (Figure 8a). DANO

DANO as an aPS is also achievable using the widely accepted

800 nm two-photon laser. Taken together, as compared to

other reported aPS materials, DANO is a high-performance

unimolecular aPS to locally generate a combination of short-

lived reactive species, paving a way to break the bottleneck of

hypoxia-limited PDT.

could be coactivated by GSH and two-photon light (t = 30 s)

irr

in a response of upconversion ﬂuorescence that showed

generation of DAPS (Figure 8b). This feature would bring

great convenience for cell studies. Actually, under two-photon

CLSM, the bright ﬂuorescence signal clearly detected in the

cells pretreated with DANO and LED light could visualize the

localization of ﬂuorogenic DAPS in cytosolic vesicles (Figure

•

Presently, careful studies on the detection and role of H in a

type I reaction mechanism for PDT remain to be explored.

c). According to the live/dead staining assay (Figure 8d),

•

Using DMPO as the H scavenger and probe, ESR evidence

tumor cells in the two-photon beam focused region could be

eradicated after very brief laser irradiation (1 or 2 min).

Therefore, DANO is perfectly compatible with two-photon

excited PDT.

•

revealed the existence of short-lived H as well as the

•

•−

•

relationship between H and O /HO . The hypothesis of

H -dependent O /HO₂^•generation pathway for organic

•

•−

dye-based type I photosensitization provides an alternative

view to look into the electron transfer process during GSH-

participating photochemical reactions. The closely linked_•

cascade free radical reaction systems, such as a cascade of H

CONCLUSIONS

In summary, we designed nonmetallic DANO as a stimuli-

responsive aPS by coupling a NO photogenerator to a light-

■

•

•−

•

•−

•

and O to form O /HO , and a cascade of O and NO to

2 2 2 2

−

•

harvesting conjugated D−A structure. The advantages of

DANO for PDT are multifold: (1) the small N(Me)−NO

substituent has a substantial photocaging eﬀect on the optical

properties of D−A motif via photoinduced electron transfer;

form ONOO , highlights the utmost position of H at the

upstream of cascade reactions. Deﬁnitely, the multilevel

synergistic interplay among ROS, RNS, RHS, and even

•

reactive sulfur species (RSS, not limited to GSH and GS )

(

2) the uncaging process is mutually controllable by GSH and

can potentially augment antitumor therapeutic outcomes.

J. Am. Chem. Soc. 2021, 143, 868−878

Journal of the American Chemical Society

https://pubs.acs.org/doi/10.1021/jacs.0c10517.

Article

per well and incubated for 24 h. After incubation with DANO (5 μM)

EXPERIMENTAL SECTION

■

in fresh growth medium, the cells were irradiated for 1 min with an

Synthetic Methods. DAPS. To a solution of 5 (150 mg, 0.2

mmol) in acetonitrile (10 mL) was added triphenylphosphine (524

mg, 2 mmol) under argon atmosphere. The reaction solution was

stirred at 85 °C for 36 h and concentrated in vacuo. Silica-gel column

chromatography using dichloromethane/methanol (10:1) as eluent

aﬀorded DAPS (184 mg, 72%) as a yellow solid; H NMR (400 MHz,

CD CN): δ 8.38 (s, 1H), 7.84−7.65 (m, 30H), 7.56 (s, 1H), 7.50 (d,

J = 4.45 Hz, 1H), 7.38 (d, J = 4.45 Hz, 1H), 6.64 (s, 1H), 5.37 (s,

−2

LED lamp (λ 405 nm, 20 mW cm ). Two-photon imaging

experiments were performed on a Leica TCS SP5 laser scanning

microscope with a 40× objective lens, using an 800 nm Ti-Sapphire

laser (∼10 W at 800 nm; ∼1% average power in the focal plane). The

upconversion emission was collected at 470−550 nm ranges.

For the two-photon excited PDT experiment, DANO-treated cells

were irradiated within a 435 × 435 μm focal area by the 800 nm TP

laser (∼100 mW) for diﬀerent scanning time (0, 60, and 120 s), and

(

H), 4.38 (m, 4H), 3.44 (m, 4H), 2.88 (s, 3 H), 2.10 (m, 4H), 1.93

incubated for additional 4 h, followed by addition of calcein-AM (1.6

m, 4H), 1.46 (s, 9H) ppm; C NMR (100 MHz, CD CN): δ 166.2,

−1

μg mL ) and PI (1.6 μg mL ) in PBS. The cells were incubated for

0 min. After washing by PBS three times, the cells were imaged by an

39.3, 147.6, 144.2, 143.6, 139.4, 135.0, 133.7, 131.3, 131.2, 130.3,

30.2, 130.1, 129.2, 127.3, 120.3, 120.0, 119.6, 117.9, 117.4, 89.9,

2.9, 72.9, 72.8, 57.3, 31.0, 30.9, 30.8, 29.6, 27.2, 21.8, 21.7, 21.4,

inverted ﬂuorescent microscope. The living and dead cells were

visualized in green and red ﬂuorescence, respectively.

1.3, and 19.3 ppm. HRMS (ESI+): m/z 558.1997 [M-2Br] ,

calculated for [C H N O P S ] : 558.1995; found 558.1997.

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

■

DANO. To a solution of DAPS (128 mg, 0.1 mmol) in 9:1 (v/v)

mixture of dichloromethane and acetic acid (10 mL) was added

NaNO (1.03 g, 15 mmol) at 0 °C. The reaction was stirred at 25 °C

for 1 h and neutralized with saturated NaHCO aqueous solution.

The mixture solution was extracted with chloroform (20 mL × 3) and

concentrated in vacuo. Silica-gel column chromatography using

CH Cl /MeOH (7:1) as eluent aﬀorded DANO (110 mg, 85%) as

Detailed materials, instrumentation, syntheses of 2 −5,

NMR spectra, and supporting ﬁgures and tables (PDF)

a yellow solid; H NMR (400 MHz, CD CN): δ 8.43 (s, 1H),8.03 (s,

AUTHOR INFORMATION

Corresponding Authors

H), 7.84−7.64 (m, 31H), 7.50 (d, J = 4.45 Hz, 1H), 7.32 (d, J = 4.45

Hz, 1H), 5.57 (s, 2H), 4.33 (m, 4H), 3.50 (s, 3H), 3.49 (m, 4H), 2.08

m, 4H),1.91 (m, 4H), 1.46 (s, 9H) ppm; ¹³C NMR (100 MHz,

CD CN): δ 166.8, 145.1, 144.5, 143.4, 135.1, 133.8, 133.7, 131.7,

■

(

Fude Feng − Department of Polymer Science & Engineering,

School of Chemistry & Chemical Engineering, Nanjing

University, Nanjing 210023, P. R. China; orcid.org/

31.1, 130.3, 130.2, 128.8, 127.8, 121.9, 120.3, 117.9, 117.4, 106.4,

3.4, 73.0, 72.6, 58.3, 31.3, 30.9, 30.7, 27.3, 21.6, 21.5, 19.4, and 19.3

000-0002-5348-5959 ; Email: fengfd@nju.edu.cn

ppm. HRMS (ESI+): m/z 572.6954 [M-2Br] , calculated for

Shu Wang − Beijing National Laboratory for Molecular

Sciences, Key Laboratory of Organic Solids, Institute of

Chemistry, Chinese Academy of Sciences, Beijing 100190, P.

R. China; orcid.org/0000-0001-8781-2535;

Email: wangshu@iccas.ac.cn

[

C H N O P S ] : 572.6946; found 572.6954.

67 65 5 5 2 2

ESR Measurements. Electron spin resonance (ESR) measure-

ments were carried out on a Bruker EMX 10/12 instrument equipped

with a TE-mode cavity. A thin-walled quartz sample tube (Bruker)

was used as the reaction vessel. The ESR settings were described as

follows: modulation frequency 100 kHz, microwave power 20 mW,

center ﬁeld 3480 G, sweep width 200 G, scan time, 2 min. Spin

trapping agents DMPO, PTIO, and TEMP were applied to identify

the relevant radical in suitable solvents. The prepared solutions were

irradiated at 298 K directly in ESR resonator using an LED lamp (405

Authors

Jian Sun − Department of Polymer Science & Engineering,

School of Chemistry & Chemical Engineering, Nanjing

University, Nanjing 210023, P. R. China

Xuetong Cai − Department of Polymer Science & Engineering,

School of Chemistry & Chemical Engineering, Nanjing

University, Nanjing 210023, P. R. China

Chengjun Wang − School of Chemistry and Chemical

Engineering, Southeast University, Nanjing 211189, P. R.

China

Ke Du − Department of Polymer Science & Engineering, School

of Chemistry & Chemical Engineering, Nanjing University,

Nanjing 210023, P. R. China

Weijian Chen − Department of Polymer Science &

Engineering, School of Chemistry & Chemical Engineering,

Nanjing University, Nanjing 210023, P. R. China; Beijing

National Laboratory for Molecular Sciences, Key Laboratory

of Organic Solids, Institute of Chemistry, Chinese Academy of

Sciences, Beijing 100190, P. R. China

−

nm, 20 mW cm ). After irradiation, the ESR spectra were recorded

and further analyzed by Bruker software Win EPR.

Computational Analysis. All calculations were performed using

the Gaussian 09 program package. The geometries of DANO and

DAPS were optimized using DFT/B3LYP/6-31G(d). The excitation

energies for the singlet and triplet excited states of DANO and DAPS

were predicted using TD-DFT/B3LYP/6-31G(d) based on the

optimized structure in acetonitrile.

Cell Viability Assays under Normoxia and Hypoxia. HeLa

cells were seeded in the 96 well plate at a density of 2 × 10 cells per

well and cultured for 24 h. The cells were treated with fresh growth

medium containing DANO (5 μM) for 2 h under 21% O or 1% O

atmosphere, followed by exposure to light irradiation for 0−5 min

−

with an LED lamp (λ 405 nm, 20 mW cm ) and incubation for an

additional 24 h in dark. After washing twice with PBS, freshly

−

prepared MTT solution (100 μL, 0.5 mg mL ) was added to each

well and incubated for 4 h. After removal of the supernatant, DMSO

(

150 μL per well) was added to dissolve the produced formazan and

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.0c10517

shaken for 10 min. The OD₄₅₀_n_mvalue of each well was recorded with

a Tecan multimode microplate reader to calculate the cell viability.

For dead/live cell costaining, the cells were treated in the same

procedure except that the MTT solution was replaced with the

Notes

The authors declare no competing ﬁnancial interest.

−

solution of Calcein-AM (1.6 μg mL ) and propidium iodide (PI, 1.6

−

μg mL ) in PBS. Then the cells were incubated for an additional 20

min. After multiple rinses with PBS, the cells were imaged by an

inverted ﬂuorescent microscope.

ACKNOWLEDGMENTS

■

We thank the National Key R&D Program of China

(2017YFA0701301, 2018YFE0200700), National Natural

Science Foundation of China (22077065, 22021002,

Two-Photon Imaging and Excited Photodynamic Therapy.

HeLa cells were seeded in 24 well plates at a density of 5 × 10 cells

J. Am. Chem. Soc. 2021, 143, 868−878

Journal of the American Chemical Society

2020102005), and Program for Changjiang Scholars and

(20) Bielski, B. H. J.; Allen, A. O. Mechanism of the

Article

disproportionation of superoxide radicals. J. Phys. Chem. 1977, 81,

048−1051.

21) Radi, R. Oxygen radicals, nitric oxide, and peroxynitrite: Redox

pathways in molecular medicine. Proc. Natl. Acad. Sci. U. S. A. 2018,

15, 5839−5848.

22) Sheng, Y.; Abreu, I. A.; Cabelli, D. E.; Maroney, M. J.; Miller,

A.; Teixeira, M.; Valentine, J. S. Superoxide Dismutases and

Superoxide Reductases. Chem. Rev. 2014 , 114 , 3854 −3918.

23) Huie, R. E.; Padmaja, S. The Reaction of NO With Superoxide.

Free Radical Res. Commun. 1993, 18, 195−199.

24) Pacher, P.; Beckman, J. S.; Liaudet, L. Nitric Oxide and

Peroxynitrite in Health and Disease. Physiol. Rev. 2007, 87, 315−424.

25) Ferrer-Sueta, G.; Campolo, N.; Trujillo, M.; Bartesaghi, S.;

Carballal, S.; Romero, N.; Alvarez, B.; Radi, R. Biochemistry of

Peroxynitrite and Protein Tyrosine Nitration. Chem. Rev. 2018, 118,

Innovative Research Team in University for ﬁnancial support.

Verma, S.; Pogue, B. W.; Hasan, T. Imaging and Photodynamic

REFERENCES

■

(

1) Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.;

(

Therapy: Mechanisms, Monitoring, and Optimization. Chem. Rev.

(

010, 110, 2795−2838.

2) Cao, H. Q.; Wang, L.; Yang, Y.; Li, J.; Qi, Y. F.; Li, Y.; Li, Y.;

(

Wang, H.; Li, J. B. An Assembled Nanocomplex for Improving both

Therapeutic Efficiency and Treatment Depth in Photodynamic

Therapy. Angew. Chem., Int. Ed. 2018, 57, 7759−7763.

Tiwari, K.; Drain, C. M. Glycosylated Porphyrins, Phthalocyanines,

(3) Lovell, J. F.; Liu, T. W.; Chen, J.; Zheng, G. Activatable

(

Photosensitizers for Imaging and Therapy. Chem. Rev. 2010, 110,

839−2857.

4) Singh, S.; Aggarwal, A.; Bhupathiraju, N. V. S. K.; Arianna, G.;

(

338−1408.

and Other Porphyrinoids for Diagnostics and Therapeutics. Chem.

Rev. 2015, 115, 10261−10306.

26) Di Mascio, P.; Martinez, G. R.; Miyamoto, S.; Ronsein, G. E.;

Medeiros, M. H. G.; Cadet, J. Singlet Molecular Oxygen Reactions

with Nucleic Acids, Lipids, and Proteins. Chem. Rev. 2019, 119,

2043−2086.

(27) Ieda, N.; Hotta, Y.; Miyata, N.; Kimura, K.; Nakagawa, H.

Photomanipulation of Vasodilation with a Blue-Light-Controllable

Nitric Oxide Releaser. J. Am. Chem. Soc. 2014 , 136 , 7085 −7091.

(28) Fraix, A.; Sortino, S. Combination of PDT photosensitizers

with NO photodononors. Photochem. Photobiol. Sci. 2018, 17, 1709−

1727.

(5) Sies, H.; Jones, D. P. Reactive oxygen species (ROS) as

pleiotropic physiological signalling agents. Nat. Rev. Mol. Cell Biol.

020, 21, 363−383.

6) Dolmans, D.; Fukumura, D.; Jain, R. K. Photodynamic therapy

for cancer. Nat. Rev. Cancer 2003, 3, 380−387.

7) Li, X.; Kolemen, S.; Yoon, J.; Akkaya, E. U. Activatable

photosensitizers: agents for selective photodynamic therapy. Adv.

Funct. Mater. 2017, 27, 1604053.

8) Wang, Y.; Liu, Y.; Sun, H.; Guo, D. Type I photodynamic

(29) Zhou, E. Y.; Knox, H. J.; Reinhardt, C. J.; Partipilo, G.; Nilges,

M. J.; Chan, J. Near-Infrared Photoactivatable Nitric Oxide Donors

with Integrated Photoacoustic Monitoring. J. Am. Chem. Soc. 2018,

140, 11686−11697.

therapy by organic −inorganic hybrid materials: From strategies to

applications. Coord. Chem. Rev. 2019, 395 , 46 −62.

9) Croce, R.; van Amerongen, H. Natural strategies for photo-

synthetic light harvesting. Nat. Chem. Biol. 2014, 10, 492−501.

(

(30) Zhang, Z.; Wu, J.; Shang, Z.; Wang, C.; Cheng, J.; Qian, X.;

Xiao, Y.; Xu, Z.; Yang, Y. Photocalibrated NO Release from N-

Nitrosated Napthalimides upon One-Photon or Two-Photon

Irradiation. Anal. Chem. 2016, 88, 7274−7280.

10) Li, M.; Xia, J.; Tian, R.; Wang, J.; Fan, J.; Du, J.; Long, S.; Song,

X.; Foley, J. W.; Peng, X. Near-Infrared Light-Initiated Molecular

Superoxide Radical Generator: Rejuvenating Photodynamic Therapy

against Hypoxic Tumors. J. Am. Chem. Soc. 2018, 140 , 14851 −14859.

(31) Patel, D. G.; Feng, F.; Ohnishi, Y.; Abboud, K. A.; Hirata, S.;

11) Li, X.; Lee, D. J.; Huang, D.; Yoon, J. Phthalocyanine-

Schanze, K. S.; Reynolds, J. R. It Takes More Than an Imine: The

Role of the Central Atom on the Electron-Accepting Ability of

Benzotriazole and Benzothiadiazole Oligomers. J. Am. Chem. Soc.

assembled nanodots as photosensitizers for highly efficient type I

photoreactions in photodynamic therapy. Angew. Chem., Int. Ed. 2018,

(

7, 9885−9890.

32) Reynolds, G. A.; Drexhage, K. H. New coumarin dyes with

012, 134, 2599−2612.

rigidized structure for flashlamp-pumped dye lasers. Opt. Commun.

975, 13, 222−225.

33) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino,

12) Nguyen, V.; Qi, S.; Kim, S.; Kwon, N.; Kim, G.; Yim, Y.; Park,

S.; Yoon, J. An Emerging Molecular Design Approach to Heavy-

Atom-Free Photosensitizers for Enhanced Photodynamic Therapy

under Hypoxia. J. Am. Chem. Soc. 2019, 141, 16243−16248.

(

(13) Sun, J.; Du, K.; Diao, J.; Cai, X.; Feng, F.; Wang, S. GSH and

Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Detection and Imaging of

Nitric Oxide with Novel Fluorescent Indicators: Diaminofluoresceins.

Anal. Chem. 1998, 70, 2446−2453.

H O Co-Activatable Mitochondria-Targeted Photodynamic Therapy

under Normoxia and Hypoxia. Angew. Chem., Int. Ed. 2020, 59,

2122−12128.

14) D ’Autrea

34) Goldstein, S.; Russo, A.; Samuni, A. Reactions of PTIO and

ux, B.; Toledano, M. B. ROS as signalling molecules:

•

−

Carboxy-PTIO with ••NO , and O . J. Biol. Chem. 2003, 278,

mechanisms that generate specificity in ROS homeostasis. Nat. Rev.

Mol. Cell Biol. 2007, 8, 813−824.

35) Hwang, C.; Sinskey, A. J.; Lodish, H. F. Oxidized redox state of

0949−50955.

glutathione in the endoplasmic reticulum. Science 1992, 257, 1496−

502.

36) Jiang, X.; Chen, J.; Bajic,

(15) Luby, B. M.; Walsh, C. D.; Zheng, G. Advanced Photosensitizer

Activation Strategies for Smarter Photodynamic Therapy Beacons.

(

Angew. Chem., Int. Ed. 2019, 58, 2558−2569.

A.; Zhang, C.; Song, X.; Carroll, S. L.;

(16) Malla, J. A.; Umesh, R. M.; Yousf, S.; Mane, S.; Sharma, S.;

Cai, Z.; Tang, M.; Xue, M.; Cheng, N.; et al. Quantitative real-time

Lahiri, M.; Talukdar, P. A Glutathione Activatable Ion Channel

Induces Apoptosis in Cancer Cells by Depleting Intracellular

Glutathione Levels. Angew. Chem., Int. Ed. 2020 , 59 , 7944 −7952.

imaging of glutathione. Nat. Commun. 2017, 8, 16087.

(37) Liu, J.; Sun, Y.; Huo, Y.; Zhang, H.; Wang, L.; Zhang, P.; Song,

D.; Shi, Y.; Guo, W. Simultaneous Fluorescence Sensing of Cys and

GSH from Different Emission Channels. J. Am. Chem. Soc. 2014, 136,

17) Sun, J.; Li, X.; Du, K.; Feng, F. A water soluble donor −

acceptor −donor conjugated oligomer as a photosensitizer for

mitochondria-targeted photodynamic therapy. Chem. Commun.

38) Okazaki, S.; Takeshita, K. Irradiation of Phenolic Compounds

74−577.

(

018, 54, 9194−9197.

with Ultraviolet Light Causes Release of Hydrated Electrons. Appl.

Magn. Reson. 2018, 49, 881−892.

(39) Bakulin, A. A.; Rao, A.; Pavelyev, V. G.; van Loosdrecht, P. H.

M.; Pshenichnikov, M. S.; Niedzialek, D.; Cornil, J.; Beljonne, D.;

Friend, R. H. The Role of Driving Energy and Delocalized States for

Charge Separation in Organic Semiconductors. Science 2012, 335,

1340−1344.

18) Zielonka, J.; Joseph, J.; Sikora, A.; Hardy, M.; Ouari, O.;

Vasquez-Vivar, J.; Cheng, G.; Lopez, M.; Kalyanaraman, B.

Mitochondria-Targeted Triphenylphosphonium-Based Compounds:

Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic

Applications. Chem. Rev. 2017 , 117 , 10043 −10120.

(19) Turrens, J. F. Superoxide Production by the Mitochondrial

Respiratory Chain. Biosci. Rep. 1997, 17, 3−8.

J. Am. Chem. Soc. 2021, 143, 868−878

Journal of the American Chemical Society

Article

40) Ou, H.; Chen, X.; Lin, L.; Fang, Y.; Wang, X. Biomimetic

(59) Makarov, N. S.; Drobizhev, M.; Rebane, A. O. Two-photon

absorption standards in the 550 −1600 nm excitation wavelength

range. Opt. Express 2008, 16, 4029−4047.

(60) Wang, S.; Wu, W.; Manghnani, P.; Xu, S.; Wang, Y.; Goh, C.

C.; Ng, L. G.; Liu, B. Polymerization-Enhanced Two-Photon

Photosensitization for Precise Photodynamic Therapy. ACS Nano

2019, 13, 3095−3105.

Donor −Acceptor Motifs in Conjugated Polymers for Promoting

Exciton Splitting and Charge Separation. Angew. Chem., Int. Ed. 2018,

(

7, 8729−8733.

41) Wang, Y.; Zhao, D.; Ji, H.; Liu, G.; Chen, C.; Ma, W.; Zhu, H.;

Zhao, J. Sonochemical Hydrogen Production Efficiently Catalyzed by

Au/TiO2. J. Phys. Chem. C 2010, 114, 17728−17733.

42) Chauvin, J.; Judee, F.; Yousfi, M.; Vicendo, P.; Merbahi, N.

Analysis of reactive oxygen and nitrogen species generated in three

liquid media by low temperature helium plasma jet. Sci. Rep. 2017, 7,

43) Capaldo, L.; Ravelli, D. Hydrogen Atom Transfer (HAT): A

562.

Versatile Strategy for Substrate Activation in Photocatalyzed Organic

Synthesis. Eur. J. Org. Chem. 2017, 2017, 2056−2071.

(44) Zhao, L.-M.; Meng, Q.-Y.; Fan, X.-B.; Ye, C.; Li, X.-B.; Chen,

B.; Ramamurthy, V.; Tung, C.-H.; Wu, L.-Z. Photocatalysis with

Quantum Dots and Visible Light: Selective and Efficient Oxidation of

Alcohols to Carbonyl Compounds through a Radical Relay Process in

Water. Angew. Chem., Int. Ed. 2017 , 56 , 3020 −3024.

45) Kobayashi, K. Pulse Radiolysis Studies for Mechanism in

Biochemical Redox Reactions. Chem. Rev. 2019, 119, 4413−4462.

46) Maroz, A.; Anderson, R. F.; Smith, R. A. J.; Murphy, M. P.

Reactivity of ubiquinone and ubiquinol with superoxide and the

hydroperoxyl radical: implications for in vivo antioxidant activity. Free

Radical Biol. Med. 2009, 46, 105−109.

(47) Tanielian, C.; Wolff, C.; Esch, M. Singlet Oxygen Production in

Water: Aggregation and Charge-Transfer Effects. J. Phys. Chem. 1996,

00, 6555−6560.

48) Xie, X.; Tang, F.; Liu, G.; Li, Y.; Su, X.; Jiao, X.; Wang, X.;

(

Tang, B. Mitochondrial Peroxynitrite Mediation of Anthracycline-

Induced Cardiotoxicity as Visualized by a Two-Photon Near-Infrared

Fluorescent Probe. Anal. Chem. 2018, 90, 11629 −11635.

Z.; Zhen, J.; Wu, S.; Ye, Z.; Zeng, J.; Huang, N.; Gu, Y. Modulating

49) Arteel, G. E.; Briviba, K.; Sies, H. Protection against

peroxynitrite. FEBS Lett. 1999, 445, 226−230.

(50) Zhao, H.; Yin, R.; Wang, Y.; Lee, Y.; Luo, T.; Zhang, J.; Qiu,

H.; Ambrose, S.; Wang, L.; Ren, J.; Yao, J.; Chen, D.; Wang, Y.; Liang,

mitochondrial morphology enhances antitumor effect of 5-ALAme-

diated photodynamic therapy both in vitro and in vivo. J. Photochem.

Photobiol., B 2017, 176, 81−91.

(51) Zhao, Z.; Chan, P.; Li, H.; Wong, K.; Wong, R. N. S.; Mak, N.;

Zhang, J.; Tam, H.; Wong, W.; Kwong, S. W.; Wong, W. Highly

Selective Mitochondria-Targeting Amphiphilic Silicon(IV) Phthalo-

cyanines with Axially Ligated Rhodamine B for Photodynamic

Therapy. Inorg. Chem. 2012, 51, 812−821.

(52) Hatz, S.; Lambert, J. D. C.; Ogilby, P. R. Measuring the lifetime

of singlet oxygen in a single cell: addressing the issue of cell viability.

Photochem. Photobiol. Sci. 2007, 6, 1106−1116.

(53) Salmeron, M. L.; Quintana-Aguiar, J.; De La Rosa, J. V.; Lopez-

Blanco, F.; Castrillo, A.; Gallardo, G.; Tabraue, C. Phenalenone-

photodynamic therapy induces apoptosis on human tumor cells

mediated by caspase-8 and p38-MAPK activation. Mol. Carcinog.

(

018, 57, 1525−1539.

54) Norman, K. G.; Canter, J. A.; Shi, M.; Milne, G. L.; Morrow, J.

D.; Sligh, J. E. Cyclosporine A suppresses keratinocyte cell death

through MPTP inhibition in a model for skin cancer in organ

transplant recipients. Mitochondrion 2010, 10, 94−101.

(55) Galluzzi, L.; Larochette, N.; Zamzami, N.; Kroemer, G.

Mitochondria as therapeutic targets for cancer chemotherapy.

Oncogene 2006, 25, 4812−4830.

(56) Buytaert, E.; Dewaele, M.; Agostinis, P. Molecular effectors of

multiple cell death pathways initiated by photodynamic therapy.

Biochim. Biophys. Acta, Rev. Cancer 2007, 1776, 86−107.

(

57) Perez-Torres, I.; Manzano-Pech, L.; Rubio-Ruíz, M. E.; Soto,

M.; Guarner-Lans, E. V. Nitrosative Stress and Its Association with

Cardiometabolic Disorders. Molecules 2020, 25, 2555.

(58) He, X.; Situ, B.; Gao, M.; Guan, S.; He, B.; Ge, X.; Li, S.; Tao,

M.; Zou, H.; Tang, B.; Zheng, L. Stereotactic Photodynamic Therapy

Using a Two-Photon AIE Photosensitizer. Small 2019, 15, 1905080.