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Saori Fujiwara, [Toru Yoshitomi](https://orcid.org/0000-0003-3847-1812), [Naoki Kawazoe](https://orcid.org/0000-0003-3916-0709), [Guoping Chen](https://orcid.org/0000-0001-6753-3678), Hiroko Bando, Hisato Hara, Hirofumi Matsui

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[Reactive oxygen species generation by photothermal effects of adhesive near-infrared agents on the plasma membrane](https://mdr.nims.go.jp/datasets/34bfcc76-0a4f-454e-9364-b7e511417c51)

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2Original articleReactive oxygen species generation by photothermal effects of adhesive near-infrared agents on the plasma membraneSaori Fujiwara1,2,†, Toru Yoshitomi1,3,4,†,*, Naoki Kawazoe1, Guoping Chen1, Hiroko Bando5, Hisato Hara6, and Hirofumi Matsui7,*1 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan2 Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan3 Department of Materials Science and Engineering, Institute of Science Tokyo, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan4 Master’s/Doctoral Program in Life Science Innovation (T-LSI), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8577, Japan5 Division of Breast and Endocrine Surgery, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan6 Department of Breast-Thyroid-Endocrine Surgery, Tsukuba Central Hospital, 1589-3　Kashiwada-cho, Ushiku, Ibaraki 300-1211, Japan7 Division of Gastroenterology, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan†Equal contribution: S.F. and T.Y. contributed equally to this work.*Corresponding authors:Toru YoshitomiResearch Center for Macromolecules and Biomaterials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, JapanE-mail: YOSHITOMI.Toru@nims.go.jp (T.Y.)Tel: +81-29-860-4739Hirofumi MatsuiDepartment of Gastroenterology, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Ibaraki 305-8575, JapanE-mail: hmatsui@md.tsukuba.ac.jp (H.M.)Tel: +81-29-853-3466Word count: 2727 wordsNumber of tables/figures: 5 figuresAuthor ContributionsConceptualization, T. Y.; methodology, S.F. and T.Y.; validation, S.F. and T.Y.; formal analysis, S.F. and T.Y.; investigation, S.F. and T.Y.; resources, T.Y., N.K., and G.C.; data curation, S.F. and T.Y.; writing—original draft preparation, S.F. and T.Y.; writing—review and editing, all coauthors; visualization, S.F. and T.Y.; supervision, T.Y.; project administration, T.Y.; funding acquisition, T.Y., N.K., G.C., and H.M. All authors have read and approved the final version of the manuscript.SummaryNear-infrared photothermal cancer therapy has attracted increasing attention due to its non-invasiveness, high selectivity, and spatiotemporally controllable local treatment. A novel near-infrared photothermal agent was developed, consisting of a plasma membrane-adhesive indocyanine green conjugated with a polycation bearing quaternary ammonium salt groups, called adhesive indocyanine green, for locally administered near-infrared photothermal cancer therapy. In this study, the photothermal effects of adhesive indocyanine green on the plasma membrane were investigated using a rat gastric mucosal cancer cell line. Upon light irradiation, adhesive indocyanine green exhibited a photothermal effect without generating singlet oxygen. Due to electrostatic interaction, adhesive indocyanine green adhered to negatively charged polysaccharides on the plasma membrane of rat gastric mucosal cancer cells. When the cells were irradiated with near-infrared light at 808 nm at 108 J/cm², levels of intracellular reactive oxygen species significantly increased despite the absence of a detectable temperature rise in the cell culture medium. These results indicate the generation of intracellular reactive oxygen species by the photothermal effects of plasma membrane-adhesive near-infrared photothermal agents.Keywords: reactive oxygen species, polycation, indocyanine green, photothermal, plasma membrane1. IntroductionIn recent years, the number of new patients with cancer and deaths from cancer has been increasing worldwide.(1) Cancer treatment mainly consists of surgery, radiation therapy, and drug therapy, which are combined in a multidisciplinary approach. Among these therapies, surgical treatment, which is a local therapy, is often very invasive, although minimally invasive treatments have been increasing in recent years. Additionally, drug-based therapies, such as chemotherapy, are sometimes limited by the patient's age or underlying disease. The development of noninvasive, localized therapies remains desirable in cancer treatment.Recently, near-infrared photothermal cancer therapy (NIR-PTCT) has received much attention due to its non-invasiveness and spatiotemporally controllable local treatment.(2-6) Through NIR light irradiation, photothermal agents convert photon energy into heat and release that heat locally to directly kill cancer cells. Recent advances in NIR light-responsive organic and inorganic materials have garnered considerable attention.(7-10) Among these materials, the use of indocyanine green (ICG), which exhibits photothermal effects, has been gaining growing interest in the field of NIR-PTCT because ICG is widely used for the identification of sentinel nodes during surgery in clinical practice.(7, 11-13) However, even intratumor administration of ICG under light irradiation fails to show high antitumor efficacy due to diffusion throughout the cells.(14) Additionally, although tumor-targeted drug delivery system using nanoparticles such as liposomes that physically encapsulate ICG are promising, their current major limitation lies in the leakage of photothermal agents, leading to insufficient efficacy.(15)Recently, we reported the development of a locally administered photothermal therapy for treating cancers using adhesive ICG (aICG) (Fig. 1).(14) aICG consists of ICG conjugated to polycation segment; thereby, completely preventing the release of ICG in vivo. In cases of breast and gastrointestinal cancers, the drugs can be locally injected under ultrasound-guided or endoscopy-guided injection.(16-18) A previous study demonstrated that NIR-PTCT with intratumor injection of aICG shows a higher antitumor effect upon light irradiation at 808 nm compared to low-molecular-weight ICG.(14) However, the photothermal effect when aICG adheres to the plasma membrane remains unclear. Therefore, in this study, we investigated the photothermal effect of aICG on the plasma membrane using a rat gastric mucosal cancer cell line (RGK1). As the plasma membrane possesses negatively charged polysaccharides on its surface, the positively charged aICG should adhere to those polysaccharides. By irradiating NIR light when aICG is attached only to the plasma membrane, the photothermal effect on the plasma membrane can be investigated.2. Materials and Methods2.1. ChemicalsICG N-hydroxysuccinimide ester (ICG-NHS) was purchased from BioActs (Incheon, Korea). Trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA), [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), and N-(3-aminopropyl)methacrylamide hydrochloride (APMAA) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Methanol, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 ham with 15mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) and sodium bicarbonate, without L-glutamine and Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham with 15mM HEPES and sodium bicarbonate, without L-glutamine and phenol red were both purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA, USA). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (WSCD-HCl) was purchased from Peptide Institute, Inc. (Osaka, Japan). 2’,7’-Dichlorodihydrofluorescein diacetate was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Singlet oxygen sensor green (SOSG) reagent was purchased from Thermo Fisher Scientific (Cleveland, OH, USA). PlasMem Bright Red and Hoechst33342 were purchased from Dojindo (Kumamoto, Japan).2.2. Preparation and characterization of PMETAC-co-PAPMAA(ICG)PMETAC-co-PAPMAA(ICG), called aICG, was synthesized according to a previously reported method.(19) The aICG was dissolved in phosphate-buffered saline (PBS) at a concentration of 2.5 mg/mL, corresponding to an ICG concentration of 13 μM. Ultraviolet-visible (UV-vis) spectra were recorded using a UV-2600 UV-visible spectrophotometer (Shimadzu Corp., Japan). Fluorescence spectra were collected using an FP-8500DS fluorescence spectrophotometer (JASCO Corp., Tokyo, Japan). The photothermal performance was evaluated using NIR laser irradiation (CivilLaser, Hangzhou, China) at an intensity of 0.9 W/cm2, and the increase in temperature was measured using an electronic thermometer (As One Corp., Tokyo, Japan). Three samples were analyzed for each quantitative measurement.2.3. Detection of singlet oxygenA total of 10 μL of SOSG (Thermo Fisher, Cleveland, OH, USA) was added to 90 µL of aICG solution (2.5 mg/mL) to achieve a final concentration of 5 µM. The mixture was irradiated with an 808 nm laser at 0.9 W/cm2 for a total fluence of 270 J/cm2 over 5 min. The fluorescence signal of SOSG was measured at an excitation/emission wavelength of 488/535 nm using a Spark multimode microplate reader (Tecan Trading AG, Switzerland).2.4. Localization of aICGRGK1 is a cell line derived from rat gastric mucosal cells (RGM1) that was transformed into a cancerous state by exposure to N-methyl-N′-nitro-N-nitrosoguanidine to become cancerous.(20) RGK1 cells were seeded onto a glass-bottom dish at a density of 3.0 × 103 cells per glass area and incubated overnight in a 5% carbon dioxide (CO₂) incubator at 37°C. After incubation, cells were washed once with cold PBS. The culture medium was replaced with 200 µL of medium without phenol red or FBS, containing the aICG (0.25 mg/mL), PlasMem Bright Red (5 μg/mL), and Hoechst33342 (5 μg/mL). At 5 min after the medium replacement, the cells were observed using a STELLARIS 8 confocal microscope (Leica Microsystems, Wetzlar, Germany). The excitation wavelengths of Hoechst33342, PlasMem Bright Red, and ICG were 405 nm, 560 nm, and 760 nm, respectively.2.5. Intracellular ROS generationRGK1 cells were seeded into a 24-well microplate at a density of 5.0 × 104 cells per well and incubated in a 5% CO2 incubator at 37°C for 2 days. 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was added at a final concentration of 1.0 × 10-7 mM, and the cells were incubated for 15 min. After incubation, cells were washed thrice with PBS to remove extracellular DCFH-DA. The medium was then replaced with 300 µL of medium (without phenol red and FBS) containing a final concentration of 0.25 mg/mL aICG. At 5 min after the medium replacement, the cells were irradiated with an 808 nm laser at 0.9 W/cm2 for 2 min (108 J/cm2) at room temperature. Following irradiation, the cells were harvested using trypsin-EDTA treatment. The fluorescence intensity within the cells was quantified using a BD Accuri™™ C6 flow cytometer (BD Biosciences San Jose, CA, USA).2.6 Evaluation of cell membrane damage and cytotoxicity RGK1 cells were seeded at a density of 3.0 × 10⁴ cells per well in a 24-well microplate and cultured for 2 d. The medium was replaced with 400 mL of medium containing aICG at a final concentration of 0.25 mg/mL. After 5 min, cells were irradiated with an 808 nm laser (0.9 W/cm²) at room temperature for 2 min (108 J/cm²), followed by staining with CytoCalcein Violet 450, Apopxin Green Indicator and 7-AAD, which are commercially available as Apoptosis/ Necrosis Assay Kit (Cat. No.: ab176749, Abcam, United Kingdom), according to product instruction. The stained cells were observed by a fluorescence microscope (BZ-710; Keyence, Osaka, Japan). Number of stained cells by CytoCalcein Violet 450, Apopxin Green Indicator, and 7-AAD was counted. The mean and SEM were calculated from three independent experiments (≧ 100 cells).2.7. Statistical analysisStatistical comparisons among more than three groups were conducted using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (GraphPad Prism, version 10); the difference was considered to be significant when p < 0.05. Statistical analysis to check the linearity was achieved with a normality Shapiro–Wilk (W) test and homoscedasticity test using GraphPad Prism.3. Results3.1.  Characterization of aICGThe chemical structure of the aICG used in this study is shown in Fig. 1. The average molecular weight of the polycation bearing quaternary ammonium salt groups was approximately 20 kDa and contained approximately 100 units of the positively charged monomer, METAC. Fig. 2a shows the absorption spectrum of aICG in PBS, which exhibited a peak at 780 nm. Fig. 2b shows the fluorescence spectrum of aICG in PBS, which displayed a peak at 800 nm.The photothermal performance of aICG at a concentration of 2.5 mg/mL (equivalent to 13 µM of ICG) was assessed under NIR laser irradiation at 808 nm for 5 min. Before irradiation, both PBS and aICG solutions were maintained at approximately 28°C. Although PBS showed no significant temperature increase after irradiation (rising to 30°C), aICG alone demonstrated a marked temperature elevation, reaching 43°C (Fig. 3a). To evaluate the generation of singlet oxygen in aICG, SOSG reagent was used to confirm the generation of singlet oxygen from aICG using laser irradiation at 808 nm.(21) SOSG emits green fluorescence upon reacting with singlet oxygen. However, no notable difference in fluorescence intensity was observed between aICG and PBS in either irradiated or non-irradiated samples (Fig. 3b). These results indicate that aICG did not generate singlet oxygen under NIR light irradiation under the present conditions.3.2.  Localization of aICGAs demonstrated in our previous study, aICG containing polycations exhibited a significantly higher therapeutic effect than low molecular weight ICG. This enhancement was attributed to the distinct cellular distribution of aICG. Although aICG is rapidly internalized and diffuses uniformly throughout the cytoplasm, aICG tends to adhere to the plasma membrane, potentially leading to enhanced cancer cell-killing efficacy. However, in previous experiments, fluorescence signals from aICG were detected in endolysosomes 24 h after treatment rather than on the plasma membrane.(14) Given that positively charged aICG can electrostatically interact with negatively charged polysaccharides on the plasma membrane, aICG should initially adhere to the plasma membrane. In this study, we first performed microscopic observations immediately after adding aICG to the cell culture medium to obtain evidence that aICG was attached to the plasma membrane. For visualization, PlasMem Bright Red and Hoechst 33242 were used to stain plasma membrane glycans and nuclei, respectively. As shown in Fig. 4, PBS-treated cells displayed membrane-localized fluorescence of PlasMem Bright Red. In aICG-treated cells, fluorescence signals from aICG clearly overlapped with those from PlasMem Bright Red, indicating that aICG rapidly adhered to the plasma membrane upon exposure. Notably, dot-like aICG fluorescence signals were observed in the cytoplasm after 15 min (data not shown), suggesting rapid internalization of aICG via endocytosis following initial membrane adherence. For comparison, the low molecular weight ICG was also added to the cell culture medium and observed under similar conditions. However, even 5 minutes after addition, it was taken up into the cells and was not localized solely to the cell membrane (Fig. S1). This is because ICG itself is a highly hydrophobic compound with its molecular weight of 774.96 g/mL and permeates the cell membrane. Therefore, it localized in the cytoplasm even at 5 min after its addition. On the other hand, aICG is a polycation with a molecular weight of about 20 kDa, which cannot permeate the cell membrane and is initially electrostatically adsorbed on negatively charged polysaccharides on the cell membrane. Since the purpose of this study was to investigate the photothermal effect of aICG that only adheres to the membrane, only aICG was used for further investigation in this study.3.3.  Intracellular ROS generationAs mentioned above, only aICG was localized on the plasma membrane after its addition to the cell culture medium. To investigate the photothermal effect on the plasma membrane, light was irradiated and the photothermal effect on the cell membrane was evaluated when aICG was attached to the cell membrane. aICG was added to the cell culture medium at a final concentration of 0.25 mg/mL, and the temperature of the cell culture medium was measured immediately after irradiation. Before NIR exposure, the average temperature of the medium was 26.3°C (Fig. 5a). Following irradiation, the temperature of the medium without and with aICG rose to 27.8 and 27.7°C, respectively, indicating no significant difference. To assess intracellular reactive oxygen species (ROS) generation, DCFH-DA was used. This probe is deacetylated by intracellular esterases to yield nonfluorescent 2', 7'-dichlorodihydrofluorescein (DCFH), which is subsequently oxidized by ROS to the highly fluorescent compound DCF. The fluorescence intensity of DCF was measured as an indicator of intracellular ROS levels. Comparisons were made between non-irradiated and irradiated RGK1 cells treated with PBS or aICG. The irradiated aICG group showed a significantly higher fluorescence intensity compared to both the non-irradiated aICG group and the irradiated PBS group (Fig. 5b). These results suggested that NIR irradiation of membrane-bound aICG induced intracellular ROS generation.3.4 Evaluation of cell membrane damage and cytotoxicity The cell membrane damage and cytotoxicity were evaluated by three fluorescence dyes: CytoCalcein Violet 450, Apopxin Green Indicator and 7-AAD. Viable cells can be stained by CytoCalcein Violet 450. Alteration in cell membrane integrity can be evaluated by 7-AAD. The translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the Apopxin Green Indicator. These staining results allow us to evaluate not only cell viability but also changes in the cell membrane due to the photothermal effect of aICG on the cell membrane. Compared to other groups, in aICG-treated and irradiated group, the proportion of cells stained with only CytoCalcein Violet 450, which is intact cells, was significantly decreased. In addition, in aICG-treated and irradiated group, the proportion of cells stained with CytoCalcein Violet 450 and 7-AAD, which means visible cells with increased cell permeability, was significantly higher than the other groups. These results indicate that approximately 80% of the cells were alive, but the permeability of the cell membrane increased in approximately 60% of the cells by the photothermal effect of aICG. This indicates evidence of aICG-induced photothermal damage of the cell membrane in RGK1 cells. Notably, although there was no significant difference, the proportion of cells stained with 7-AAD and Apopxin Green Indicator tended to be increased. This result suggests that aICG-induced photothermal effect caused the exposure of phosphatidylserine to the cell surface, leading to apoptosis in approximately 20% of the cells.4. DiscussionaICG was developed for locally administered NIR-PTCT, where light can be directly delivered to tumor tissue using an optical fiber, and a photothermal agent can be administered locally within or near the tumor site.(14, 22) Subsequent light irradiation to tumor tissue leads to cancer cell death. Although low molecular weight photothermal agents such as ICG can increase the temperature, these agents fail to raise the temperature locally, limiting their therapeutic efficacy.(14) In contrast, aICG adheres to the plasma membrane, contributing to its higher anti-cancer effect.(14) Previous experiments revealed the accumulation of aICG in lysosomes, likely due to translocation via the endocytic pathway after final adhesion to plasma membranes through electrostatic interactions.(14)In this study, to investigate the photothermal effect of aICG on the plasma membrane, light was irradiated onto aICG before it entered the endosome through endocytosis, and the production of intracellular ROS was examined. As shown in Fig. 4, aICG was detected only at the plasma membrane and not within lysosomes. Light irradiation under these conditions allowed for the evaluation of effects caused specifically by temperature increases at the plasma membrane. Upon light exposure, intracellular ROS levels increased significantly despite no observable increase in the temperature of the surrounding medium. These results demonstrate that localized heating at the plasma membrane can enhance intracellular ROS production. Here, change in the cell membrane due to the photothermal effect of aICG attached to the cell membrane were confirmed. As shown in Fig. 6, the aICG-treated group upon light irradiation significantly increased in plasma membrane permeability, which was detected by staining with 7-AAD. This proves that aICG attached to the cell membrane raises the temperature near the cell membrane, causing changes in the structure of the cell membrane. Furthermore, although no significant difference was observed, there was a tendency for phosphatidylserine to be exposed to the cell surface. This also suggests that the temperature increase in the cell membrane caused by aICG reduces the stability of the cell membrane, causing phosphatidylserine in the inner membrane to migrate to the outer membrane. These results demonstrate that increasing the temperature near the cell membrane by aICG causes changes in the membrane. As there has so far been no technology that can selectively increase the temperature near the cell membrane, the mechanism by which intracellular ROS increases due to structural changes in the cell membrane is still unknown. In future, we would like to elucidate its mechanism. In addition, after adhering to the cell membrane, aICG is taken up into the cell by endocytosis, and then, part of aICG reaches lysosomes. Therefore, by considering the timing of light irradiation, it would be possible to improve the anti-cancer effect of aICG.In conclusion, this study demonstrated that aICGs, when attached to cell membranes through electrostatic interactions, significantly increased intracellular ROS following NIR light irradiation despite the absence of a temperature increase in the surrounding cell culture medium. These findings indicate that a local temperature rise at the cell membrane induces intracellular ROS. Furthermore, the results suggest that photothermal agents can generate intracellular oxidative stress even when localized on the cell membrane rather than internalized. Although mechanistic analysis of these phenomena is needed, aICG holds promise as a novel photothermal agent for topically administered NIR-PTCT, offering a new strategy for inducing intracellular oxidative stress.Conflict of InterestThe authors declare no conflicts of interest.AcknowledgmentsThis study was partially supported by the Japan Society for the Promotion of Science KAKENHI, Grant Number 21H03813 (awarded to T.Y., G.C., N.K., and H.M.).References1 Global Burden of Disease Cancer C, Kocarnik JM, Compton K, et al. Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life Years for 29 Cancer Groups From 2010 to 2019: A Systematic Analysis for the Global Burden of Disease Study 2019. JAMA Oncol 2022; 8: 420-444.2 Li J, Zhang W, Ji W, et al. Near infrared photothermal conversion materials: mechanism, preparation, and photothermal cancer therapy applications. J Mater Chem B 2021; 9: 7909-7926.3 Sutrisno L, Chen H, Yoshitomi T, Kawazoe N, Yang Y, Chen G. PLGA-collagen-BPNS Bifunctional composite mesh for photothermal therapy of melanoma and skin tissue engineering. J Mater Chem B 2022; 10: 204-213.4 Sutrisno L, Chen H, Chen Y, et al. Composite scaffolds of black phosphorus nanosheets and gelatin with controlled pore structures for photothermal cancer therapy and adipose tissue engineering. Biomaterials 2021; 275: 120923.5 Sun J, Zhao H, Xu W, Jiang GQ. Recent advances in photothermal therapy-based multifunctional nanoplatforms for breast cancer. Front Chem 2022; 10: 1024177.6 Alamdari SG, Amini M, Jalilzadeh N, et al. Recent advances in nanoparticle-based photothermal therapy for breast cancer. Journal of Controlled Release 2022; 349: 269-303.7 Jung HS, Verwilst P, Sharma A, Shin J, Sessler JL, Kim JS. Organic molecule-based photothermal agents: an expanding photothermal therapy universe. 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Biomater Sci 2019; 7: 3158-3164.14 Fujiwara S, Yoshitomi T, Hoshi A, et al. Locally Administered Photothermal Therapy for Breast Cancer using Endolysosome-Targeted Indocyanine Green Conjugated with Polycation. Journal of Surgery and Research 2024; 07: 122-133.15 Yoon HJ, Lee HS, Lim JY, Park JH. Liposomal Indocyanine Green for Enhanced Photothermal Therapy. ACS Appl Mater Interfaces 2017; 9: 5683-5691.16 Kooistra B, Wauters C, Strobbe L, Wobbes T. Preoperative cytological and histological diagnosis of breast lesions: A critical review. Eur J Surg Oncol 2010; 36: 934-940.17 Bhatt AA, Whaley DH, Lee CU. Ultrasound-Guided Breast Biopsies: Basic and New Techniques. J Ultrasound Med 2021; 40: 1427-1443.18 Akagi T, Yasuda K, Tajima M, et al. Sodium alginate as an ideal submucosal injection material for endoscopic submucosal resection: preliminary experimental and clinical study. Gastrointestinal Endoscopy 2011; 74: 1026-1032.19 Komatsu Y, Yoshitomi T, Furuya K, et al. Long-Term Fluorescent Tissue Marking Using Tissue-Adhesive Porphyrin with Polycations Consisting of Quaternary Ammonium Salt Groups. Int J Mol Sci 2022; 23.20 Shimokawa O, Matsui H, Nagano Y, et al. Neoplastic transformation and induction of H+,K+ -adenosine triphosphatase by N-methyl-N'-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line. In Vitro Cell Dev Biol Anim 2008; 44: 26-30.21 Liu HH, Carter PJH, Laan AC, Eelkema R, Denkova AG. Singlet Oxygen Sensor Green is not a Suitable Probe for O-1(2) in the Presence of Ionizing Radiation. Scientific Reports 2019; 9.22 Komatsu Y, Yoshitomi T, Doan VTH, et al. Locally Administered Photodynamic Therapy for Cancer Using Nano-Adhesive Photosensitizer. Pharmaceutics 2023; 15.Figure legendsFig. 1. Chemical structure of aICG. A copolymer of poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] and poly[N-(3-aminopropyl)methacrylamide hydrochloride] conjugated with indocyanine green [PMETAC-co-PAPMAA(ICG)] is referred to as aICG. In this structure, indocyanine green is conjugated to a polycation bearing quaternary ammonium salt groups. The average molecular weight of the polycation was approximately 20 kDa and contained approximately 100 units of the positively charged monomer, 2-(methacryloyloxy)ethyltrimethylammonium chloride. aICG, adhesive indocyanine green.Fig. 2. Characterization of aICG. (a) Ultraviolet-visible absorption spectrum of aICG in PBS. (b) The fluorescence emission spectrum of aICG in PBS (excitation at 760 nm). aICG was dissolved in PBS at a concentration of 2.5 mg/mL, corresponding to 13 μM of ICG. aICG, adhesive indocyanine green; PBS, phosphate-buffered saline.Fig. 3. Photothermal and photodynamic properties of aICG. (a) Temperature changes of aICG solution (2.5 mg/mL) upon NIR light irradiation at 808 nm (0.9 W/cm2) for 5 min (270 J/cm2). (b) Singlet oxygen generation by aICG under NIR light irradiation was detected using the singlet oxygen-sensitive dye SOSG. The fluorescence intensity of SOSG was measured after irradiation at 808 nm (0.9 W/cm2) for 5 min (270 J/cm2). (-), without light irradiation; (+), with light irradiation. Data were expressed as means ± standard deviation (n=3). aICG, adhesive indocyanine green; NIR, near-infrared; SOSG, singlet oxygen sensor green.Fig. 4. Intracellular localization of aICG in RGK1 cells. Upper images: fluorescence signal of aICG.Middle images: Plasmem Red staining of the plasma membrane. Lower images: Hoechst 33342 staining of cell nuclei. aICG was used at a concentration of 0.25 mg/mL, corresponding to 1.3 μM ICG. aICG, adhesive indocyanine green; RGK1, rat gastric mucosal cancer cell line.Fig. 5. Effects of NIR light irradiation on aICG-treated cells. (A) Temperature of the cell culture medium after light irradiation. Data were shown as mean ± SEM (n = 3). * p < 0.05 compared to other groups. (B) ROS generation in aICG-treated RGK1 cells was detected using DCFH-DA. Median fluorescence intensity (arbitrary units, a.u.) was measured in cells treated with PBS or aICG, with or without NIR light irradiation. Data were shown as mean ± SEM (n = 3). * p < 0.05 compared to other groups. NIR, near-infrared; aICG, adhesive indocyanine green; PBS, phosphate-buffered saline; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; SEM, standard error of the mean.Fig. 6. Alteration in cell membrane of aICG-treated cells upon light irradiation. (A) Microscopic observation of the cells stained by CytoCalcein Violet 450, 7-AAD and Apopxin Green Indicator. Living cells were stained by CytoCalcein Violet 450. In cells with increased cell membrane permeability, 7-AAD translocates into the nucleus and emits red fluorescence. The cells with exposure of phosphatidylserine to the cell surface are stained by Apopxin Green Indicator. (B) The percentage of cells stained by CytoCalcein Violet 450, 7-AAD, and Apopxin Green Indicator (green). Number of stained cells by CytoCalcein Violet 450, Apopxin Green Indicator, and 7-AAD was counted. The mean and SEM were calculated from three independent experiments (≧ 100 cells).Fig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.image3.tifimage4.tifimage5.tifimage6.tiffimage1.tifimage2.tif