Synthesis and biological evaluation of EGFR binding peptides for near-infrared photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that involves photoimmunotherapy drug injection and NIR light exposure. In NIR-PIT, antibodies are commonly used as target-directed molecules carrying IRDye700DX (IR700). However, antibodies have disadvantages, such as high cost, complex development strategies, and poor tumor penetration. In contrast, peptides have lower production costs, can be easy to chemically synthesize and modify, and can also be used for tumor-targeting like antibodies. In this study, we developed a novel PIT drug using a peptide as the target-directed molecule. Epidermal growth factor receptor (EGFR) was selected as the target, and monovalent and bivalent EGFR-binding peptides were synthesized. The bivalent peptide showed sufficient binding to EGFR-positive cells, and a bivalent peptide-IR700 conjugate with a long linker induced morphological changes in EGFR-positive cells. Additionally, the drug significantly reduced cell viability in vitro in an NIR light-dose- and drug-concentration-dependent manner. These results indicate the feasibility of NIR-PIT in treating cancer using peptide-based drugs.

Theoretical Design and Synthesis of Caged Compounds Using X-Ray-Triggered Azo Bond Cleavage.

Caged compounds are frequently used in life science research. However, the light used to activate them is commonly absorbed and scattered by biological materials, limiting their use to basic research in cells or small animals. In contrast, hard X-rays exhibit high bio-permeability due to the difficulty of interacting with biological molecules. With the main goal of developing X-ray activatable caged compounds, azo compounds are designed and synthesized with a positive charge and long π-conjugated system to increase the reaction efficiency with hydrated electrons. The azo bonds in the designed compounds are selectively cleaved by X-ray, and the fluorescent substance Diethyl Rhodamine is released. Based on the results of experiments and quantum chemical calculations, azo bond cleavage is assumed to occur via a two-step process: a two-electron reduction of the azo bond followed by N─N bond cleavage. Cellular experiments also demonstrate that the azo bonds can be cleaved intracellularly. Thus, caged compounds that can be activated by an azo bond cleavage reaction promoted by X-ray are successfully generated.

In vivo imaging of acute physiological responses after treatment of cancer with near-infrared photoimmunotherapy.

PURPOSE: Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer phototherapy using an antibody-photosensitizer conjugate (Ab-IR700). By NIR light irradiation, Ab-IR700 forms a water-insoluble aggregation on the plasma membrane of cancer cells, leading to lethal membrane damage of cancer cells with high selectivity. However, IR700 produces singlet oxygen, which induces non-selective inflammatory responses such as edema in normal tissues around the tumor. Understanding such treatment-emergent responses is important to minimize side effects and improve clinical outcomes. Thus, in this study, we evaluated physiological responses during NIR-PIT by magnetic resonance imaging (MRI) and positron emission tomography (PET). PROCEDURES: Ab-IR700 was intravenously injected into tumor-bearing mice with two tumors on the right and left sides of the dorsum. At 24 h after injection, a tumor was irradiated with NIR light. Edema formation was examined by T1/T2/diffusion-weighted MRI and inflammation was investigated by PET with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). Because inflammation can increase vascular permeability via inflammatory mediators, we evaluated changes in oxygen levels in tumors using a hypoxia imaging probe, [18F]fluoromisonidazole ([18F]FMISO). RESULTS: The uptake of [18F]FDG in the irradiated tumor was significantly decreased compared to the control tumor, indicating the impairment of glucose metabolism induced by NIR-PIT. MRI and [18F]FDG-PET images showed that inflammatory edema with [18F]FDG accumulation was present in the surrounding normal tissues of the irradiated tumor. Furthermore, [18F]FMISO accumulation in the center of the irradiated tumor was relatively low, indicating the enhancement of oxygen supply due to increased vascular permeability. In contrast, high [18F]FMISO accumulation was observed in the peripheral region, indicating enhancement of hypoxia in the region. This could be because inflammatory edema was formed in the surrounding normal tissues, which blocked blood flow to the tumor. CONCLUSIONS: We successfully monitored inflammatory edema and changes in oxygen levels during NIR-PIT. Our findings on the acute physiological responses after light irradiation will help to develop effective measures to minimize the side effects in NIR-PIT.

[Research on Photoimmunotherapy Based on Photochemical Property of Molecules].

Photoimmunotherapy (PIT) is a new cancer therapy that uses near-infrared (NIR) light and a conjugate of an antibody and a photosensitizer (IR700). Since both NIR light and the conjugate are not toxic for human, PIT has attracted attention as a promising cancer therapy with less side effects. However, there is no photosensitizer for PIT other than IR700. To improve the therapeutic effect, more light-sensitive dye is needed. To this end, we have studied the cytotoxic mechanism of PIT, showing that the hydrophilic axial ligand cleavage of IR700 by NIR light irradiation is important for the cytotoxicity. Herein, I focused on the triplet state (T1) of IR700 because the light-induced axial ligand cleavage reaction is thought to occur via the T1. First, the quantum yield of intersystem crossing, which is the transition efficiency from the excited singlet state (S1) to T1, was determined by analysis of the T1 kinetics using fluorescence correlation spectroscopy (FCS). Also, I examined whether the cytotoxicity of IR700 can be changed in the presence of a triplet quencher. The findings obtained here will be important information for the design of a new photosensitizer for PIT in the future.

Ligand release from silicon phthalocyanine dyes triggered by X-ray irradiation.

Ligand release from silicon phthalocyanine (SiPc) dyes triggered by near-infrared (NIR) light is a key photochemical reaction involving caged compounds based on SiPc. Although NIR light is relatively permeable compared with visible light, this light can be attenuated by tissue absorption and scattering; therefore, using light to induce photochemical reactions deep inside the body is difficult. Herein, because X-rays are highly permeable and can produce radicals through the radiolysis of water, we investigated whether the axial ligands of SiPcs can be cleaved using X-ray irradiation. SiPcs with different axial ligands (alkoxy, siloxy, oxycarbonyl, and phenoxy groups) were irradiated with X-rays under hypoxic conditions. We found that the axial ligands were cleaved via reactions with hydrated electrons (e-aq), not OH radicals, generated from water in response to X-ray irradiation, and SiPc with alkoxy groups exhibited the highest cleavage efficiency. A quantitative investigation revealed that X-ray-induced axial ligand cleavage proceeds via a radical chain reaction. The reaction is expected to be applicable to the molecular design of X-ray-activatable functional molecules in the future.

Correction: Takakura et al. In Vitro and In Vivo Cell Uptake of a Cell-Penetrating Peptide Conjugated with Fluorescent Dyes Having Different Chemical Properties. Cancers 2021, 13, 2245.

There was an error in the description in the original publication, and it contained information that should not have been disclosed [...].

Reduction of tumor hypoxia by anti-PD-1 therapy assessed using pimonidazole and [18F]FMISO.

INTRODUCTION: Hypoxia is common in solid tumors and creates an immunosuppressive environment that leads to resistance to immunotherapy, such as an anti-programmed death receptor-1 (PD-1) therapy. It has been suggested that anti-PD-1 therapy may reduce tumor hypoxia by remodeling the tumor vasculature; however, it is unclear how anti-PD-1 therapy reduces hypoxia over time. Therefore, we investigated the relationship between hypoxia and immune activation by anti-PD-1 therapy in murine cancer models. METHODS: Anti-PD-1 antibody was injected to CT26- and MC38-tumor-bearing mice on days 0 and 5. Tumor hypoxia was non-invasively evaluated using positron emission tomography (PET) with [18F]fluoromisonidazole ([18F]FMISO) on days 3 and 7. Histological analysis was conducted to investigate the infiltration of immune cells in [18F]FMISO-accumulated hypoxic area. In addition, the immune cell population in tumors and the percentages of cancer and immune cells under hypoxic conditions were analyzed at single-cell level using flow cytometry. RESULTS: Flow cytometric analysis of CT26 tumors on day 3 showed that anti-PD-1 therapy reduced hypoxia without inhibition of tumor growth. In addition, the infiltration of CD8+ T cells was increased in treated tumors. In contrast to CT26 tumors, the percentage of hypoxic cells in MC38 tumors did not change on days 3 and 7, and there was minimal immune activation induced by anti-PD-1 antibody. Changes in hypoxia in CT26 tumors were not detected by [18F]FMISO-PET, but autoradiogram showed that [18F]FMISO accumulated in immunosuppressed areas, where the infiltration of immune cells was relatively low. CONCLUSION: Reduction of hypoxia was induced in CT26 tumor, in which adequate immune response to anti-PD-1 therapy was exhibited, at an early time point before suppression of tumor growth. Our findings suggest that anti-PD-1 therapy can create a tumor microenvironment that facilitates immune activation by reducing hypoxia.

Development of Novel 191Pt-Labeled Hoechst33258: 191Pt Is More Suitable than 111In for Targeting DNA.

This study aims to establish new labeling methods for no-carrier-added radio-Pt (191Pt) and to evaluate the in vitro properties of 191Pt-labeled agents compared with those of agents labeled with the common emitter 111In. 191Pt was complexed with the DNA-targeting dye Hoechst33258 via diethylenetriaminepentaacetic acid (DTPA) or the sulfur-containing amino acid cysteine (Cys). The intranuclear fractions of 191Pt- and 111In-labeled Hoechst33258 were comparable, indicating that the labeling for 191Pt via DTPA or Cys and the labeling for 111In via DTPA worked equally well. 191Pt showed a DNA-binding/cellular uptake ratio of more than 1 order of magnitude greater than that of 111In. [191Pt]Pt-Hoechst33258 labeled via Cys showed a higher cellular uptake than that labeled via DTPA, resulting in a very high DNA-binding fraction of [191Pt]Pt-Cys-Hoechst33258 and extensive DNA damage. Our labeling methods of radio-Pt, especially via Cys, promote the development of radio-Pt-based agents for use in Auger electron therapy targeting DNA.

Electron Donors Rather Than Reactive Oxygen Species Needed for Therapeutic Photochemical Reaction of Near-Infrared Photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) employs molecularly targeted antibodies conjugated with a photoabsorbing silicon-phthalocyanine dye derivative which binds to cancer cells. Application of NIR light following binding of the antibody-photoabsorber conjugates (APCs) results in ligand release on the dye, dramatic changes in solubility of the APC-antigen complex, and rapid, irreversible cell membrane damage of cancer cells in a highly selective manner, resulting in a highly immunogenic cell death. Clinically, this process results in edema after treatment mediated by reactive oxygen species (ROS). Based on the chemical and biological mechanism of NIR-PIT cytotoxicity and edema formation, in order to minimize acute inflammatory edema without compromising therapeutic effects, l-sodium ascorbate (l-NaAA) was administered to quench harmful ROS and accelerate the ligand release reaction. l-NaAA suppressed acute edema by reducing ROS after NIR-PIT yet did not alter the therapeutic effects. NIR-PIT could be performed safely under existence of l-NaAA without side effects caused by unnecessary ROS production.

Comparison of low-molecular-weight ligand and whole antibody in prostate-specific membrane antigen targeted near-infrared photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a cancer phototherapy that uses antibody-IR700 conjugate (Ab-IR700) and NIR light. Ab-IR700 forms aggregates on the plasma membranes of targeted cancer cells after light exposure, inducing lethal physical damage within the membrane. Low-molecular-weight (LMW) ligands are candidate targeting moieties instead of antibodies, but whether LMW-IR700 conjugates induce cell death by aggregation, the same mechanism as Ab-IR700, is unknown. Thus, we investigated differences in cytotoxicity and mechanisms between LMW-IR700 and Ab-IR700 targeting prostate-specific membrane antigen (PSMA). Both conjugates decreased cell viability to the same degree after light irradiation, but different morphological changes were observed in PSMA-positive LNCaP cells by microscopy. Cell swelling and bleb formation were induced by Ab-IR700, but only swelling was observed in cells treated with LMW-IR700, suggesting the cells were damaged via different cytotoxic mechanisms. However, LMW-IR700 induced bleb formation, a hallmark of NIR-PIT with Ab-IR700, when singlet oxygen was quenched or LMW-IR700 was localized only on the plasma membrane. Moreover, the water-soluble axial ligands of LMW-IR700 were cleaved, consistent with previous reports on Ab-IR700. Thus, the main cytotoxic mechanisms of Ab-IR700 and LMW-IR700 differ, although LMW-IR700 on the plasma membrane can cause aggregation-mediated cytotoxicity as well as Ab-IR700.

In Vitro and In Vivo Cell Uptake of a Cell-Penetrating Peptide Conjugated with Fluorescent Dyes Having Different Chemical Properties.

In molecular imaging, a targeting strategy with ligands is widely used because specificity can be significantly improved. In fluorescence imaging based on a targeting strategy, the fluorescent dyes conjugated with ligands may affect the targeting efficiency depending on the chemical properties. Herein, we used a cell-penetrating peptide (CPP) as a ligand with a variety of fluorescent cyanine dye. We investigated in vitro and in vivo cell uptake of the dye-CPP conjugates when cyanine dyes with differing charge and hydrophilicity/lipophilicity were used. The results showed that the conjugates with positively charged and lipophilic cyanine dyes accumulated in cancer cells in vitro, but there was almost no accumulation in tumors in vivo. On the other hand, the conjugates with negatively charged and hydrophilic cyanine dyes did not accumulate in cancer cells in vitro, but fluorescence was observed in tumors in vivo. These results show that there are some cases in which the cell uptake of the dye-peptide conjugates may differ significantly between in vitro and in vivo experiments due to the chemical properties of the fluorescent dyes. This suggests that attention should be paid to the chemical properties of fluorescent dyes in fluorescence imaging based on a targeting strategy.

Photoimmunotherapy: A new cancer treatment using photochemical reactions.

Photoimmunotherapy (PIT) is a new molecular-targeted phototherapy in which administration of an antibody conjugated to IR700 (Ab-IR700, a phthalocyanine derivative) is followed by irradiation with near-infrared light. PIT induces cell death due to cell membrane damage, and the formation of IR700 aggregates on the cell membrane triggered by photochemical reactions is an important mechanism of cell killing. Specifically, water-soluble axial ligands of IR700 are cleaved by the photochemical reaction, and the phthalocyanine stacks up due to the π-π interaction, resulting in the formation of aggregates. In addition, the formation of IR700 radical anions and their protonation are essential for the progress of this photochemical reaction. The elucidation of these mechanisms may lead to the development of more effective compounds in the future. In addition, the optical properties of phthalocyanine are expected to expand the medical application of phthalocyanine derivatives in the future.

Molecular Design of d-Luciferin-Based Bioluminescence and 1,2-Dioxetane-Based Chemiluminescence Substrates for Altered Output Wavelength and Detecting Various Molecules.

Optical imaging including fluorescence and luminescence is the most popular method for the in vivo imaging in mice. Luminescence imaging is considered to be superior to fluorescence imaging due to the lack of both autofluorescence and the scattering of excitation light. To date, various luciferin analogs and bioluminescence probes have been developed for deep tissue and molecular imaging. Recently, chemiluminescence probes have been developed based on a 1,2-dioxetane scaffold. In this review, the accumulated findings of numerous studies and the design strategies of bioluminescence and chemiluminescence imaging reagents are summarized.

Theoretical and Experimental Studies on the Near-Infrared Photoreaction Mechanism of a Silicon Phthalocyanine Photoimmunotherapy Dye: Photoinduced Hydrolysis by Radical Anion Generation.

Invited for this month's cover are the collaborating groups of Dr. Masato Kobayashi and Prof. Mikako Ogawa, both from Hokkaido University, Sapporo, Japan. The cover picture shows the photochemical reaction process of the near-infrared (NIR) photoimmunotherapy dye IR700, and subsequent cancer cell death. A computational study predicted that ligand dissociation, which is known to initiate cancer cell death, proceeds by the hydrolysis of the IR700 radical anion, rather than as a direct result of NIR irradiation. This mechanism has also been supported by experimental work. Read the full text of the Communication at 10.1002/cplu.202000338.

Theoretical and Experimental Studies on the Near-Infrared Photoreaction Mechanism of a Silicon Phthalocyanine Photoimmunotherapy Dye: Photoinduced Hydrolysis by Radical Anion Generation.

Ligand release from IR700, a silicon phthalocyanine dye used in near-infrared (NIR) photoimmunotherapy, initiates cancer cell death after NIR absorption, although its photochemical mechanism has remained unclear. This theoretical study reveals that the direct Si-ligand dissociation by NIR light is difficult to activate because of the high dissociation energy even in excited states, i. e., >1.30 eV. Instead, irradiation generates the IR700 radical anion, leading to acid-base reactions with nearby water molecules (i. e., calculated pKb for the radical anion is 7.7) to produce hydrophobic ligand-released dyes. This suggests two possibilities: (1) water molecules participate in ligand release and (2) light is not required for Si-ligand dissociation as formation of the IR700 radical anion is sufficient. Experimental evidence confirmed possibility (1) by using 18 O-labeled water as the solvent, while (2) is supported by the pH dependence of ligand exchange, providing a complete description of the Si-ligand bond dissociation mechanism.

Surface Modification of Liposomes Using IR700 Enables Efficient Controlled Contents Release Triggered by Near-IR Light.

Stimuli-responsive liposomes are promising drug carriers for cancer treatment because they enable controlled drug release and the maintenance of desired drug concentrations in tumor tissue. In particular, near-IR (NIR) light is a useful stimulus for triggering drug release from liposomes based on its advantages such as deep tissue penetration and safety. Previously, we found that a silicon phthalocyanine derivative, IR700, conjugated to antibodies, can induce the rupture of the cell membrane following irradiation by NIR light. Based on this finding, we constructed IR700-modified liposomes (IR700 liposomes) and evaluated their drug release properties triggered by NIR light. IR700 liposomes released substantial amounts of encapsulated calcein following irradiation by NIR light. Drug release was substantially suppressed by the addition of sodium azide, suggesting that liposomal membrane permeabilization was mediated by singlet oxygen generated from IR700. Moreover, calcein release from IR700 liposomes triggered by NIR light was promoted under conditions of deoxygenation and the presence of electron donors. Thus, membrane disruption should be induced by the physical change of IR700 from highly hydrophilic to hydrophobic as we previously described, although singlet oxygen can cause a certain level of membrane disruption under normoxia. We also observed that doxorubicin-encapsulated IR700 liposomes exhibited significant cytotoxic effects against CT-26 murine colon carcinoma cells following NIR light exposure. These results indicate that IR700 liposomes can efficiently release anti-cancer drugs following NIR light irradiation even under hypoxic conditions and, therefore, they would be useful for cancer treatment.

Accumulation of hypoxia imaging probe "18F-FMISO" in macrophages depends on macrophage polarization in addition to hypoxic state.

OBJECTIVE: Macrophages play an essential role in immune response, and are closely related to the progression of diseases such as cancer and atherosclerosis. Macrophages polarize to M1 or M2 type, which is related to the environmental hypoxic state. Previously, we found that 18F-FMISO uptake varied according to expression levels of biomolecules such as glutathione S-transferase P1 (GST-P1), which catalyzes the conjugation of glutathione to 18F-FMISO metabolites, and multidrug resistance-associated protein 1 (MRP1), which exports glutathione-18F-FMISO metabolite conjugates out of cells. However, the relationship between macrophage polarization and 18F-FMISO accumulation remains unclear. METHODS: Mouse peritoneal macrophages were polarized to either the M1 or M2 type, and were treated with 18F-FMISO. Then, their radioactivity after a 4 h incubation period under normoxic (21% O2) or hypoxic (1% O2) condition was measured. GST-P1 and MRP1 expression levels were measured by qRT-PCR. RESULTS: M2 macrophages exhibited a significantly higher uptake of 18F-FMISO than non-polarized (M0) macrophages, whereas M1 macrophages had a significantly lower uptake than M0 macrophages (M0: 1.05 ± 0.22, M1: 0.34 ± 0.02, M2: 4.17 ± 0.36 %dose/mg protein). The GST-P1 expression level in M1 macrophages was higher than that in M2 and M0 macrophages [GST-P1/ß-actin normalized by M0: 9.0 ± 3.7 (M1), 1.2 ± 0.2 (M2)]. The MRP1 expression level in M1 macrophages was significantly higher than that in M2 and M0 macrophages [MRP1/ß-actin normalized by M0 macrophages: 5.1 ± 2.1 (M1), 2.8 ± 1.0 (M2)]. CONCLUSIONS: 18F-FMISO accumulation in macrophages may depend on the polarization state in addition to hypoxic condition.

Photoinduced Ligand Release from a Silicon Phthalocyanine Dye Conjugated with Monoclonal Antibodies: A Mechanism of Cancer Cell Cytotoxicity after Near-Infrared Photoimmunotherapy.

Photochemical reactions can dramatically alter physical characteristics of reacted molecules. In this study, we demonstrate that near-infrared (NIR) light induces an axial ligand-releasing reaction, which dramatically alters hydrophilicity of a silicon phthalocyanine derivative (IR700) dye leading to a change in the shape of the conjugate and its propensity to aggregate in aqueous solution. This photochemical reaction is proposed as a major mechanism of cell death induced by NIR photoimmunotherapy (NIR-PIT), which was recently developed as a molecularly targeted cancer therapy. Once the antibody-IR700 conjugate is bound to its target, activation by NIR light causes physical changes in the shape of antibody antigen complexes that are thought to induce physical stress within the cellular membrane leading to increases in transmembrane water flow that eventually lead to cell bursting and necrotic cell death.

Anti PD-1 treatment increases [18F]FDG uptake by cancer cells in a mouse B16F10 melanoma model.

BACKGROUND: Programmed cell death 1 (PD-1) inhibitors act as immune checkpoint inhibitors and are more effective for improving survival time with less toxicity as compared with conventional chemotherapies. In anti PD-1 therapy, it is important to evaluate metabolism in the cancer microenvironment, as this helps to clarify the pathological conditions. Herein, we investigate the early effects of PD-1 therapy on 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake in vivo, focusing on cell distribution and glycolysis in both cancer and immune cells. RESULTS: In a B16F10 melanoma model, [18F]FDG-positron emission tomography (PET) was performed before treatment and 7 days after the start of treatment. Values were calculated as the percentage-injected activity per gram of tissue (%IA/g). Flow-cytometry was then performed to assess immune cell populations and glucose metabolism. There was a negligible difference in [18F]FDG uptake between tumors in the treatment group and non-treatment group before the treatment. In contrast, mean [18F]FDG uptake in the treatment group tumors was significantly higher (8.06 ± 0.48 %IA/g; P = 0.0074) than that in the non-treatment group (4.02 ± 1.03 %IA/g) after anti PD-1 treatment. Assessment of tumor immune cell populations showed that treatment slightly enriched CD8+ T cells and CD4+ T cells; however, infiltration of immune cells was negligible, and thus, immune cells were not responsible for the increase in [18F]FDG uptake. On the other hand, anti PD-1 treatment significantly increased glucose transporter 1 (GLUT1) and hexokinase II expression in CD45- cancer cells, indicating that anti PD-1 treatment increased glucose metabolism in cancer cells. CONCLUSION: The present study shows that anti PD-1 therapy increases glucose metabolism in cancer cells.

Changes in plasma membrane damage inducing cell death after treatment with near-infrared photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer phototherapy modality using an antibody conjugated to a photosensitizer, IRDye700DX. When the conjugate binds to the plasma membrane and is exposed to NIR light, NIR-PIT-treated cells undergo swelling, and target-selective necrotic/immunogenic cell death is induced. However, the cytotoxic mechanism of NIR-PIT has not been elucidated. In order to understand the mechanism, it is important to elucidate how the damage to the plasma membrane induced by NIR light irradiation changes over time. Thus, in the present study, we investigated the changes in plasma membrane permeability using ions and molecules of various sizes. Na+ flowed into cells immediately after NIR light irradiation, even when the function of transporters or channels was blocked. Subsequently, fluorescent molecules larger than Na+ entered the cells, but the damage was not large enough for dextran to pass through at early time points. To assess these phenomena quantitatively, membrane permeability was estimated using radiolabeled ions and molecules: 111 InCl3 , 111 In-DTPA, and 3 H-H2 O, and comparable results were obtained. Although minute plasma membrane perforations usually do not induce cell death, our results suggest that the minute damage induced by NIR-PIT was irreversibly extended with time. In conclusion, minute plasma membrane damage is a trigger for the increase in plasma membrane permeability, cell swelling, and necrotic/immunogenic cell death in NIR-PIT. Our findings provide new insight into the cytotoxic mechanism of NIR-PIT.