Comparative Evaluation of Wavelength-Dependent Photodynamic Therapy Efficacy Using Representative Red and Near-Infrared Photosensitizers in a Single Tumor Model

Abstract

Introduction

This study aims to investigate and fairly compare the oncological therapeutic efficacy of red photodynamic therapy (Red-PDT) and near-infrared photodynamic therapy (NIR-PDT), to support the selection of suitable photosensitizers (PSs) for optimal PDT.

Methods

Two different representative PSs, trastuzumab-HiLyte Fluor™ 647 conjugate (Tra-HLF647) and trastuzumab-Indocyanine Green conjugate (Tra-ICG), activated by two laser systems at 635 nm and 808 nm, respectively, were used. To ensure a fair comparison, we used the same A4 cell line/tumor model expressing the same target, human epidermal growth factor receptor 2 (HER-2), and employed the same delivery approach. To comprehensively evaluate and compare the potential effects of Tra-HLF647-mediated Red-PDT and Tra-ICG-mediated NIR-PDT, we conducted cell viability imaging assays, intracellular reactive oxygen species (ROS) generation measurements, longitudinal monitoring of tumor volume changes, histological and immunohistochemical (IHC) analyses of tumor sections, and measurements of tumor necrotic depth.

Results

Both PDTs exerted similar rapid cell death in cell viability imaging assays. There was no significant difference in ROS generation between cells subjected to Red-PDT and NIR-PDT. Both PDTs caused a statistically significant tumor growth delay compared to the control groups; however, no significant difference was detected between the Red-PDT and NIR-PDT groups. The H&E-stained sections of tumors that received Red-PDT and NIR-PDT showed a similar pattern of necrosis-associated features. No conspicuous tissue damage was observed in the control groups. The depth of necrosis, estimated via the coincided accumulation of a fluorescent necrosis marker (AF546-pHLIP) and utilized as an indirect index to approximate laser light penetration, was also nearly identical between tumors treated with Red-PDT and NIR-PDT.

Conclusions

Target-specific Red-PDT and NIR-PDT, using their respective PSs, demonstrated equivalent therapeutic efficacy in tumor models. These findings suggest that wavelength differences between Red-PS and NIR-PS may not critically impact treatment outcomes, offering flexibility in fluorophore selection for future PS conjugate design.

Introduction

Cancer remains a life-threatening global disease with significant societal, public health, and economic impacts. According to GLOBOCAN 2022 estimates, there were nearly 20 million new cancer cases and 9.7 million cancer deaths in 2022. By 2050, the global cancer burden is projected to rise to 35 million new cases, an increase of 77% from 2022 levels. ¹

In oncology, various conventional anticancer therapies, such as surgery, chemotherapy, radiotherapy, and immunotherapy, have been developed. However, these approaches may impose physical and psychological burdens on cancer patients. ² As an alternative, phototherapy (PT), encompassing photodynamic therapy (PDT), photothermal therapy (PTT), and photoimmunotherapy (PIT), has garnered renewed attention due to its advantages such as minimal invasiveness, repeatability, cost-effectiveness, targeted action, and improved therapeutic comfort for patients. ³

PDT is an anticancer modality that requires a photosensitizer (PS) and light to kill cancer cells. Upon irradiation with light of the appropriate wavelength, a PS absorbs the light energy and interacts with biomolecules to generate cytotoxic free radicals and reactive oxygen species (ROS) or converts ground-state oxygen (³O₂) to highly reactive, cytotoxic excited-state singlet oxygen (¹O₂).^4,5 These are very toxic to cells as they instantly oxidize important biomolecules such as proteins and nucleic acids (DNA, RNA). Abundant ROS and ¹O₂ can directly damage cells and/or vasculature, causing cancer cell death through necrosis and/or apoptosis, and inducing cooperative effects of inflammatory and immune responses.^4,6

The comprehensive efficiency of PDT depends on several factors, including the characteristics of activating light, in situ dosimetry, the PS's ability to preferentially accumulate in targeted tumors, and the tumor's location and microenvironment.^7,8 Among the multiple factors, the excitation wavelength of PS and the wavelength of applied light are major considerations in PDT. As light travels through tissue, it is absorbed by various biomolecules, resulting in attenuation. ⁹ Photon energy is inversely proportional to the wavelength. For example, according to wavelength-to-energy calculations, the photon energies of red light (700 nm) and NIR light (800 nm) are approximately 1.77 eV and 1.55 eV, respectively.

Recent advances in PSs and light sources are encouraging for the future progress of PDT. ¹⁰ PDT has evolved through three generations of PSs. First-generation PSs, such as hematoporphyrin derivatives, were limited by poor tumor selectivity, shallow tissue penetration, and prolonged photosensitivity.^10–12 Second-generation PSs offered better photophysical properties but still limited tumor selectivity.^11–13 Third-generation PSs have emerged, incorporating tumor-targeting ligands (eg, antibodies, peptides) or nanocarrier-based delivery systems to enhance selectivity and reduce systemic toxicity.^14–15 Among these, Indocyanine Green (ICG) has gained renewed attention due to its NIR absorption (∼800 nm), established clinical safety, and suitability for image-guided therapy. ¹⁶ Lately, PDT was preferred to be performed in red to NIR wavelengths range (>620 nm) since longer wavelength light penetrates deeper into tissues than short wavelength light (UV, blue, green, yellow), increasing the likelihood of damaging tumor cells. Conventional PSs typically absorb visible light (400-700 nm) and suffer from poor penetration depth. ¹⁷ In general, the PDT window is reported to be about 650–900 nm, ¹⁷ and PSs activated at 600–800 nm are commonly used. ¹⁸ Red light (600-700 nm) absorbing PSs such as aminolaevulinic acid (Ex: 635 nm), temoporfin (Ex: 652 nm), and talaporfin (Ex: 664 nm), have been developed over decades for clinical applications or trials.^4,11,12 We also previously reported a new PS, folate-Si-rhodamine-1 (Ex: 652 nm), that could potentially be used for folate receptor-targeted PDT in preclinical cancer models. ¹⁹

Meanwhile, NIR light (700-1000 nm) absorbing PSs such as palladium bacteriopheophorbide (Tookad) (Ex: 762 nm) and bacteriochlorin (Redaporfin) (Ex: 749 nm) ¹¹ in clinical studies, as well as indocyanine green (ICG) (Ex: 774 nm)^20–23 in preclinical studies, have also been reported for cancer-targeted PDT. However, there have been no studies that properly compare the efficacy of Red-PDT and NIR-PDT. A comparative evaluation of the efficacy in in vitro and in vivo models can aid in selecting appropriate fluorophores for designing new PS conjugates, predicting the potential of PS, optimizing treatment protocols, and reducing the risk of failure in later stages of clinical trials.

The purpose of this study is to compare the phototherapeutic efficacy of Red-PDT and NIR-PDT for cancer treatment. To ensure an unbiased comparison between Red-PS and NIR-PS activated by their respective excitation wavelengths, it is crucial to use the same cell line/tumor model expressing the same target molecule and to apply an identical delivery approach for the PSs. To achieve this objective, we conducted PDT using a consistent cell line/tumor model and target-specific PSs, created by conjugating a cancer-targeting antibody to appropriate fluorophores, followed by appropriate laser light irradiation at equivalent doses (Figure 1).

Figure 1.

Schematic Illustration of the Experimental Design and Methodology Used to Ensure a Fair Comparison of Therapeutic Efficacy Between Red Photodynamic Therapy (Red-PDT) and Near-Infrared Photodynamic Therapy (NIR-PDT) in Cancer Treatment.

Materials and Methods

Chemicals and Reagents

The human monoclonal antibody anti-human epidermal growth factor receptor 2 (HER-2) (Trastuzumab) was purchased from Chugai Pharmaceutical Co., Ltd (Tokyo, Japan). HiLyte Fluor^TM 647 (HLF647) Labeling Kit-NH2 and Indocyanine (ICG) Labeling Kit-NH2 were purchased from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA). ReadyProbes^TM cell viability imaging kit for cell viability imaging assays and CellROX^® Oxidative Stress Green Reagent to measure the reactive oxygen species (ROS) in live cells were purchased from Thermo Fisher Scientific K.K., (Tokyo, Japan). The pH (low) insertion peptides (pHLIPs) labeled with the fluorescent dye Alexa Fluor^TM 546 C5 (AF546, Ex/Em: 556/573 nm) maleimide (Thermo Fisher Scientific K.K.,), namely AF546-pHLIP construct, was produced and purchased from Scrum Inc. (Tokyo, Japan).

Cell Line

The A4 cell line, an immortalized mouse fibroblast cell line established from mouse 3T3 cells and transfected with HER-2-expression vector, was cultured and maintained in RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan), 100 U/mL penicillin-G sodium, and 100 μg/mL streptomycin sulfate (Invitrogen, Carlsbad, CA, USA) at 37 ◦C in a humidified atmosphere containing 5% CO₂.

Animal and Tumor Model

All animal experiments were conducted in accordance with protocols approved by the institutional guidelines of the Animal Care and Use Committee of the National Institutes for Quantum Science and Technology (QST; Protocol No. 07−1064−29, Chiba, Japan, approved on March 17, 2023). Male BALB/cAJcl-nu/nu mice (4 weeks old) were obtained from CLEA Japan (Shizuoka, Japan) and housed under controlled conditions (23 °C, 50% relative humidity, 12-h light/dark cycle) with ad libitum access to water and autofluorescence-free food. Mice were acclimatized for one week prior to experimentation. Xenograft tumor models were established by subcutaneous inoculation of early-passage A4 cells (2 × 10⁶ cells/site, all in 100 μL medium) into both femoral regions of nude mice. All animal experimental procedures were performed under 2.5% isoflurane inhalation anesthesia. To minimize animal use while ensuring reproducibility and statistical relevance, the minimum number of mice was used across all experiments. Sample sizes were determined based on prior experience with tumor growth variability and reference to similar published studies. While no formal power analysis was performed, group sizes were considered adequate for detecting anticipated differences in therapeutic efficacy. Tumor-bearing mice were randomly assigned to experimental groups by manual randomization to reduce selection bias.

For fluorescence imaging studies evaluating biodistribution and tumor-specific accumulation of PS conjugates (Tra-HLF, Tra-ICG), tumor-bearing mice (n = 3 mice; 6 tumors per group), were manually randomized into two experimental groups (Tra-HLF group, Tra-ICG group). For in vivo PDT efficacy evaluation, additional tumor-bearing mice were randomly assigned to four groups: untreated control, PS injection only, laser light exposure only, and PDT. Each group consisted of 4 mice bearing bilateral tumors (8 tumors per group), totaling 16 mice and 32 tumors per study. To assess in vivo and ex vivo tumor fluorescence intensity (FI) before and after PDT, as well as for immunohistochemical and histological analyses, one additional representative mouse were included per group (n = 1 mouse; 2 tumors per mouse). To assess PDT-induced tumor necrosis depth, further mice were treated with Red-PDT or NIR-PDT, and tumors (n = 3 tumors per group) were harvested for histological analysis. No animals were lost during the experiments. At the study endpoint, mice were euthanized by cervical dislocation under isoflurane anesthesia, and tumor tissues were collected for analysis. The study adheres to the ARRIVE 2.0 guidelines for reporting animal research. ²⁴

Synthesis of Trastuzumab-HiLyte Fluor™ 647 (Tra-HLF) and Trastuzumab-Indocyanine Green (Tra-ICG)

Trastuzumab was first dissolved in distilled water. Conjugation with HLF647 and ICG was performed using the HLF647 Labeling Kit and ICG Labeling Kit, respectively, following the manufacturer's protocols. Both NH2-reactive HLF647 and NH2-reactive ICG, contain a succinimidyl ester group, which can easily form a covalent bond with the amino group of Trastuzumab without any activation. Briefly, NH2-reactive dyes were mixed with a Trastuzumab (150 μg) solution on the membrane of a filtration tube and incubated at 37 ◦C for 10 min. After incubation, buffer solution was added, and the mixture was centrifuged. The resulting conjugates, Tra-HLF (Ex/Em: 655/670 nm) and Tra-ICG (Ex/Em: 774/805 nm), were recovered by pipetting with phosphate-buffered saline (PBS). The concentration of the conjugates was determined using the Bradford protein assay with a Bio-Rad microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The absorbance of Tra-HLF and Tra-ICG were measured at 655 nm and 800 nm, respectively, using a SpectraMax iD3 microplate reader (Molecular Devices Japan K.K, Tokyo, Japan). The dyes-to-antibody ratios were calculated to be approximately 0.4 for both Tra-HLF and Tra-ICG.

Fluorescence Microscopy Studies

A4 cells (5 × 10⁴ cells/well) were seeded onto an EZVIEW glass bottom culture plate (24 well, black; IWAKI, Shizuoka, Japan) in complete RPMI 1640 medium (1 mL) and incubated for 24 h at 37 °C. The medium was then replaced with fresh medium containing either Tra-HLF (5 μg/500 μL medium) or Tra-ICG (5 μg/500 μL medium), followed by overnight incubation at 37 ˚C. After incubation, the medium was discarded, and the cells were washed and replenished with phenol red-free RPMI 1640 medium (Wako). Fluorescence imaging was performed using a Keyence BZ-X810 fluorescence microscope (Keyence Japan Co., Ltd, Osaka, Japan). For Tra-HLF accumulation, the Cyanine 5 (Cy5) filter set (Ex: 620/60 nm, Em: 700/75 nm, dichroic mirror: 660 nm) was used. For Tra-ICG accumulation, the Cyanine 7 (Cy7) filter set (Ex: 710/75 nm, Em: 810/90 nm, dichroic mirror: 760 nm) was used. Appropriate exposure times were used consistently. Bright-field images were also acquired.

Cell Viability Imaging Assay

As described above, A4 cells (5 × 10⁴ cells/well) were seeded onto an EZVIEW glass bottom culture plate (24 well, black; IWAKI) and incubated overnight at 37 °C. The following day, the culture medium was replaced with fresh medium containing either Tra-HLF (5 μg/500 μL medium) or Tra-ICG (5 μg/500 μL medium), or medium without conjugates (control). After overnight incubation, the medium was discarded. Cells treated with Tra-HLF were subsequently irradiated with red laser light (635 ± 3 nm) using a Red Diode Fiber Output Laser system (Laser Create Co., Tokyo, Japan). Cells incubated with Tra-ICG were irradiated with NIR laser light (808 ± 3 nm) using an Infrared Diode Fiber Output Laser system (Laser Create Co.) For both lasers, the fluence was 6.4 J/cm², achieved by delivering a total power of 0.1 W for 50 s over an irradiation area of 0.785 cm². The beam diameter at the aperture is approximately 5 х 6 mm in both laser systems. The distance between the cell layer and the light source was set to 3 cm. The irradiation dose was measured using a Starlite thermal laser power sensor and optical power meter (OPHIR Japan, Saitama, Japan). Following laser light exposure, each well was refilled with phenol red-free RPMI 1640 medium (Wako) (500 μL) containing two staining reagents: NucBlue^® Live reagent and NucGreen^® Dead reagent (Thermo Fisher Scientific K.K,). Cells were incubated at 37 °C for 30 min. Nuclei of viable cells and dead cells were stained with NucBlue^® Live reagent and NucGreen^® Dead reagent, respectively. Fluorescence images were acquired 1.5 h after laser light exposure using a Keyence BZ-X810 microscope (Keyence Japan Co., Ltd). The DAPI filter set (Ex/Em: 360/460 nm) was used for NucBlue^®, and the GFP filter set (Ex/Em: 475/509 nm) was used for NucGreen^®. Bright-field images were also acquired.

Measurement of Reactive Oxygen Species (ROS) Generation in Cells

To assess ROS generation mediated by PDT, cell preparation and laser light exposure were performed as described above. Following irradiation, each well was refilled with the phenol red-free RPMI 1640 medium (Wako) (500 μL) containing CellROX^® Oxidative Stress Green Reagent (Ex/Em: 485/520 nm) (5 μM) and incubated at 37 °C for 30 min. Cell images were acquired with a Keyence BZ-X810 microscope (Keyence Japan Co, Ltd) with GFP filter sets for CellROX^® Green (Ex/Em: 470/525 nm) at 1.5 h after laser light exposure. CellROX^® Green is a cell-permeant, DNA-binding dye that fluoresces strongly upon oxidation, with signals primarily localized to the nucleus and mitochondria. Image acquisitions were performed using consistent exposure settings across all samples. Quantitative analysis was carried out using the Hybrid Cell Count application of BZ-X810 Analyzer software (Keyence Japan Co, Ltd). After setting the threshold, cell separation, and black balance in analysis, four quantitative metrics were measured and compared between samples: luminescent cell counts (cells exhibiting ROS signal), total luminescent area (cumulative ROS-positive area), total luminance (overall ROS-associated signal intensity), and average luminance (mean signal intensity per unit area). The ROS measurement experiment was performed once for each sample, covering four treatment conditions per PDT modality. While the single-run design limited the ability to perform statistical inference, the analysis provided indicative comparative trends in ROS generation among experimental groups.

In Vivo Fluorescence Imaging for Biodistribution and Tumor-Specific Accumulation of PS Conjugates

One week after inoculation, subcutaneous A4 tumors in mice reached approximately 8 mm in the longest diameter. Tumor-bearing mice (n = 3 mice, 6 tumors per group) received intravenous injection of Tra-HLF (100 μg/100 μL PBS) via the tail vein. To assess the biodistribution and clearance of the conjugates, mice were anesthetized with 2.5% isoflurane inhalation, and in vivo NIR fluorescence imaging was performed. Fluorescence images of the dorsal position of mice were acquired using the VISQUE™ InVivo Smart-LF imaging system (Vieworks Co., Ltd, Gyeonggi, Korea) set with Cy5.5 filter sets (Ex/Em: 630-680/690-740 nm) and constant imaging parameters (exposure time: 100 ms, binning: 1 × 1, light intensity: high, mode: low gain). Similarly, tumor-bearing mice injected with Tra-ICG (100 μg in 100 μL PBS) were imaged using the same system with ICG filter sets (Ex/Em: 740-805/810-860 nm) and consistent imaging parameters (exposure time: 300 ms, binning: 1 × 1, light intensity: high, mode: low gain). Longitudinal imaging was performed at pre-injection and multiple post-injection time points: 5 min, 15 min, 30 min, and 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 168 h. For imaging at 24, 48, 72, 96, and 168 h, the exposure time was set to 500 ms.

In Vivo Photodynamic Therapy (PDT) of Tumors

Subcutaneous tumors reached an average maximum length of approximately 8 mm by day 7 post-xenografting. Tumor-bearing mice (n = 4 mice, 8 tumors per group) were randomly assigned to four experimental groups. Red-PDT and NIR-PDT were performed with the respective specified light sources on two consecutive days following intravenous injection of either Tra-HLF647 or Tra-ICG. For Red-PDT, mice received Tra-HLF647 (100 μg in 100 μL PBS) via tail vein injection. Tumors were irradiated at 24- and 48-h post-injection using a 635 ± 3 nm diode laser (Laser Create Co.). A fluence of 63.7 J/cm² was delivered by applying a total power of 0.25 W for 200 s over an irradiation area of 0.785 cm². For NIR-PDT, mice were administered Tra-ICG (100 μg in 100 μL PBS) and irradiated under the same conditions using an 808 ± 3 nm diode laser. The distance between the tumor surface and the laser source was maintained at 3 cm, and the rest of the mouse body was shielded with aluminum foil to prevent off-target light exposure. The fiber end output diameter was ∼10 mm, and the entire tumor was irradiated at once using a fixed laser source for a duration of 200 s. Separate cohorts of tumor-bearing mice were used for the Red-PDT and NIR-PDT studies. Each study included four experimental groups, with each group consisting of 4 mice, totaling 16 mice and 32 tumors per study. In both studies, the experimental groups were as follows: Group 1 received no treatment; group 2 received PS injection only; group 3 received laser light exposure only on two consecutive days (24 h and 48 h); and group 4 received PS injection followed by laser light exposure at 24 and 48 h after injection. This experimental design ensured consistent and comparative evaluation of treatment efficacy across both PDT modalities. Tumor volumes (n = 8 per group) were measured twice a week for 13 days using calipers. Tumor volume was calculated according to the formula: volume (mm³) = [length (mm)] × [width (mm)]² × 0.5. To evaluate treatment response, relative tumor volume was calculated as the ratio of tumor volume on each indicated day to the baseline volume prior to treatment. Mouse body weight was also recorded twice weekly, and general health and skin condition at the irradiation site were monitored daily.

Immunohistochemical and Histological Analysis

Four hours after the second light exposure, one mouse from each group was sacrificed for tissue analysis. The tumors were excised, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm-thick slices. Serial tissue sections were fixed on glass slides and stained with hematoxylin and eosin (H&E) to assess histopathological changes. In addition, serial tumor sections were rehydrated and subjected to antigen retrieval for immunohistochemical (IHC) analysis. For IHC, tumor sections were stained with an anti-human Ki-67 polyclonal antibody (Dako Denmark, Glostrup, Denmark) at a 1:200 dilution, following previously described protocols. ²⁵ Ki-67 was used as a marker for cellular proliferation. ²⁶ All stained slides were examined using an Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan).

Examination of Tissue Destruction Induced by PDT

To assess tissue damage induced by PDT, AF546-pHLIP (10 nmol) was intravenously administered 2 h after the second laser light exposure. Two hours after administration, mice were sacrificed, tumors were excised and vertically bisected through the center of the predominantly light-exposed area. Bisected tumor blocks were subsequently frozen, and cryo-sectioned into serial sections (6 μm). Sections were stored in the dark at −80 ◦C until further procedures. The representative section was imaged using the Keyence BZ-X810 fluorescence microscopy system (Keyence) with TRITC filter sets (Ex: 545/25, Em: 605/70) to capture the whole-section images of AF546-pHLIP. Bright-field images were also acquired (data not shown). After acquiring a fluorescence image, the same section was stained with H&E. Fluorescence and H&E images were then registered and merged using Adobe Photoshop (version 25.7.0.504; Adobe Inc., San Jose, CA, USA) to correlate necrotic regions with AF546-pHLIP accumulation.

Statistical Analysis

The quantitative data were presented as the mean ± standard deviation (SD). Relative Tumor volume differences between groups were determined using two-factor ANOVA with replication (Microsoft Excel, Redmond, WA, United States), followed by Tukey's test. Comparisons of relative tumor volume and necrotic depth between the Red-PDT and NIR-PDT groups were performed using unpaired, two-tailed Student's t-tests (Microsoft Excel). A P value < 0.01 was considered statistically significant.

Results

Absorbance Spectra of Tra-HLF647 and Tra-ICG, and Their Accumulation in A4 Cells

The conjugations of Trastuzumab to the PSs, HLF647 and ICG, were summarized and illustrated (Figure 2A). The absorbance spectra of the resulting conjugates, Tra-HLF647 and Tra-ICG, which exhibited characteristic peaks at 650 nm and 800 nm, respectively, were examined and confirmed (Figure 2B). Fluorescence microscopy revealed that both conjugates accumulated in A4 cells, with fluorescence signals localized to the cell membrane and intracellular compartments, indicating preferential and target-specific binding to HER-2 (Figure 2C).

Figure 2.

Synthesis and Characterization of Tra-HLF and Tra-ICG. (A) Conjugation of the Fluorophores HLF647 and Indocyanine Green (ICG) to the Anti-HER-2 Antibody (Trastuzumab) Was Performed Using a Commercial Labeling Kit, According to the Manufacturer's Instructions. (B) Absorbance Spectra of Tra-HLF (Red-PS) and Tra-ICG (NIR-PS), Showing Peak Absorbance at 650 and 800 nm, Respectively. (C) Fluorescence Microscopy Images of HER2-Expressing A4 Cells Incubated With Tra-HLF or Tra-ICG for 24 h, Visualized Using Appropriate Filter Sets. Intense Membranous and Intracellular Fluorescence Signals Indicate Substantial Binding and Internalization of Both PS Conjugates in A4 Cells. (Scale Bar = 50 μm; 40× Objective Magnification).

Phototoxic Cell Death Induced by Red-PDT and NIR-PDT

Under fluorescence microscopy, rapid cell death induced by Red-PDT or NIR-PDT was visualized as green-colored cells stained with the NucGreen^® Dead reagent, while the nuclei of viable cells were stained blue with the NucBlue^® Live reagent (Figures 3A, 3B). The extent of phototoxic cell death induced by Red-PDT and NIR-PDT was similar. In contrast, noticeable phototoxic cell death was not observed with Tra-HLF treatment alone, red laser light exposure alone, Tra-ICG treatment alone, NIR laser light exposure alone, or no treatment (Figures 3A, 3B).

Figure 3.

Assessment of Phototoxicity in HER2-Expressing A4 Cells Using a Cell Viability Imaging Assay 1.5 h After Photodynamic Therapy (PDT). Cells Were Treated With Anti-HER2 Photosensitizer (PS) Conjugates, Tra-HLF or Tra-ICG, Followed by Irradiation With Red (Red-PDT) or Near-Infrared (NIR-PDT) Laser Light. Cell Viability Was Assessed Using NucBlue® Live Reagent (Blue: Viable Nuclei) and NucGreen® Dead (Green: Nuclei of Dead Cells) Reagent. (A) Red-PDT Induced Rapid and Extensive Cell Death, as Evidenced by Strong Green Nuclear Staining. (B) A Comparable Level of Phototoxicity Was Observed Following NIR-PDT. In Contrast, Control Groups Treated With PS Alone or Laser Light Exposure Alone Showed Minimal Cytotoxicity, Confirming That Cell Death Was PDT-Specific. (Scale Bar = 100 μm; 10× Objective Magnification).

Reactive Oxygen Species (ROS) Generation Induced by Red-PDT and NIR-PDT

Intracellular ROS generation was assessed using CellROX^® Green, which fluoresces upon oxidation. The luminescence signal which correlates with intracellular ROS, was visualized, quantified, and compared across different cell samples. Under fluorescence microscopy, the ROS generation induced by Red-PDT and NIR-PDT was evidenced by higher signal intensity—reflected by an increased number of luminescent cells, larger total luminescent area, and higher total and average luminance—compared to the samples treated with Tra-HLF alone, red laser light alone, Tra-ICG alone, NIR laser light alone, or left untreated (Figures 4A, 4B, and 4C). However, no obvious difference in ROS generation was observed between the cell samples subjected to Red-PDT and NIR-PDT, suggesting that both modalities induce similar oxidative stress in cells. (Figures 4A, 4B, and 4C).

Figure 4.

Measurement and Quantitative Analysis of Intracellular ROS Generation Following Photodynamic Therapy (PDT). HER2-Expressing A4 Cells Were Incubated With Tra-HLF or Tra-ICG, Followed by Irradiation With Red (Red-PDT) or Near-Infrared (NIR-PDT) Laser Light. Intracellular ROS Levels Were Detected Using a ROS-Sensitive Fluorescent Dye (CellROX® Green), and Fluorescence Images Were Acquired 1.5 Hours Post-Irradiation. (A) Red-PDT Induced a Marked Increase in ROS Generation, Visualized as Green Fluorescence. Quantification Was Performed Using Four Metrics: Number of Luminescent Cells (Cells Exhibiting ROS Signal), Total Luminescent Area (Cumulative ROS-Positive Area), Total Luminance (Overall ROS-Associated Signal Intensity), and Average Luminance (Mean Signal Intensity per Unit Area). These Metrics Reflect Both the Spatial Extent and Intensity of ROS Production at the Cellular Level, Enabling a More Comprehensive Assessment of PDT-Induced Oxidative Stress. (B) NIR-PDT Similarly Induced Elevated Intracellular ROS Generation Across All Metrics. However, No Significant Difference in ROS Generation Was Found Between Cells Treated With Red-PDT and Those Treated With NIR-PDT. (Scale Bar = 100 μm; 20× Objective Magnification). Note: Due to the Single-Run Design of the ROS Measurement Experiment, Statistical Inference Is Limited. However, the Findings Provide Representative Comparative Trends in ROS Generation Among the Experimental Groups.

Tumor-Specific Accumulation of PS Conjugates in Small Animals

Longitudinal fluorescence imaging of representative mice following injection of Tra-HLF647 and Tra-ICG revealed the time-dependent biodistribution and tumor-specific accumulation of the antibody–photosensitizer (PS) conjugates. Both conjugates gradually and specifically accumulated in tumors and were excreted from the body. Tumor accumulation was detectable within a few hours post-injection, peaked at 24–48 h, and persisted to some extent up to 168 h (Figure 5A, 5D). Concurrently, clearance from non-target tissues was observed over time. The PDT performed 24 h after PS injection resulted in a noticeable decrease in tumor FI within 30 min post-PDT in representative mice (Figures 5B, 5E). To further evaluate biodistribution, representative mice were euthanized immediately after PDT, and tumors along with major organs were excised for ex vivo fluorescence imaging. Tumors exhibited higher FI than major organs, confirming selective accumulation. Notably, tumors subjected to PDT showed reduced FI compared to those that received PS only, corroborating the in vivo imaging results (Figures 5C, 5F).

Figure 5.

Time-Dependent Fluorescence Imaging of Photosensitizer (PS) Distribution and Tumor Accumulation in a Representative A4 Tumor-Bearing Mouse. (A, D) Serial In Vivo Fluorescence Imaging Was Performed Before and After Intravenous Injection of Tra-HLF or Tra-ICG, Respectively. Imaging Time Points Included: 5 Min, 15 Min, 30 Min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 168 h Post-Injection. Progressive PS Distribution and Tumor-Specific Accumulation Were Observed Over Time, Indicating Effective Targeting and Retention. (B, E) Fluorescence Intensity (FI) in Tumors Was Compared Before and 30 Minutes After Photodynamic Therapy (PDT). A Reduction in Tumor FI Was Observed Post-PDT, Suggesting Partial Photobleaching of the PS. (C, F) Ex Vivo Fluorescence Imaging of Excised Tumors and Major Organs Immediately After PDT Revealed Higher FI in Tumors Compared to Non-Target Organs, Confirming Selective Accumulation. Tumors Treated With PDT Showed Reduced FI Relative to Those Treated With PS Alone, Providing Further Evidence of PDT-Induced Photobleaching.

Phototherapeutic Effects of Red-PDT and NIR-PDT on A4 Tumors in Nude Mice

Tumor-bearing mice were treated according to the schedules illustrated in Figure 6A. Both Red-PDT and NIR-PDT groups exhibited similar patterns of tumor growth inhibition. Statistically significant reductions in relative tumor volume were observed in both PDT groups compared to the control groups (PS only, light exposure only, and no treatment), with p < 0.01 (Figure 6B). However, no significant difference was detected between the Red-PDT and NIR-PDT groups. Ex vivo analysis of tumors excised on day 13 post-PDT showed smaller tumor sizes in the PDT groups relative to the controls (Figure 6C). No body weight loss was observed in any group (Figure 6D), and no signs of skin damage or adverse effects on general health were detected in the mice.

Figure 6.

Phototherapeutic Effects of Red Photodynamic Therapy (Red-PDT) and Near-Infrared Photodynamic Therapy (NIR-PDT) on A4 Xenograft Tumors. (A) Schematic of the Experimental Treatment Protocol. Tumor-Bearing Mice Were Randomly Assigned to Four Groups (n = 8 Tumors per Group): Untreated Control, Photosensitizer (PS) Injection Alone, Laser Light Exposure Alone, and Photodynamic Therapy (PDT). (B) Tumor Growth Was Monitored Over Time and Expressed as the Relative Tumor Volume. The Left Graph Shows Results for Red-PDT and the Right for NIR-PDT. Tumors Treated With PDT Exhibited Significantly Delayed Growth Compared to All Control Groups. Statistical Analysis Was Performed Using ANOVA. Data Are Presented as Mean ± SD (n = 8 Tumors per Group). *P < 0.01 for Red-PDT vs. Other Groups; *P < 0.01 for NIR-PDT vs. Other Groups. No Statistically Significant Difference in Relative Tumor Volume Was Observed Between Red-PDT and NIR-PDT Groups (Student's t-Test). (C) Ex Vivo Images of Tumors Excised 13 Days Post-Treatment Were Shown. The Left Panel Shows Tumors From the Red-PDT Experiment and the Right From the NIR-PDT Experiment. (Scale Bar = 10.04 mm). (D) Average Body Weight of Mice Throughout the Study Period Was Shown in the Left Graph (Red-PDT) and Right Graph (NIR-PDT). No Significant Differences Were Observed Among the Groups, Indicating Minimal Systemic Toxicity.

Evaluation of the Effect of PDT on Tumor Tissue by Histological Examination and IHC Analysis

Histological examination of H&E-stained tumor sections revealed hallmark features of necrosis and apoptosis in tumors treated with either Red-PDT or NIR-PDT. These included chromatin condensation (pyknosis), nuclear fragmentation (karyorrhexis), nuclear fading (karyolysis), and the presence of anuclear necrotic cells with a glossy, homogenous eosinophilic appearance (Figure 7A, 7B). These necrosis-associated features were consistently observed in both treatment groups. In contrast, no discernible histological damage was observed in tumors from the control groups, including those treated with PS only, light exposure only, or left untreated (Figures 7A, 7B). IHC analysis further supported these findings. Tumor regions subjected to PDT exhibited weak nuclear staining for Ki-67, a marker of cellular proliferation, and showed a marked reduction in the number of Ki-67-positive cells, indicating effective suppression of tumor cell proliferation by both Red-PDT and NIR-PDT (Supplementary Figure 1).

Figure 7.

Histological Examination of Tumor Tissue Damage Following Red-PDT and NIR-PDT in A4 Xenograft Models. Hematoxylin and Eosin (H&E) Staining Was Performed on Tumor Sections Collected on Day 2, Four Hours After the Second Laser Light Exposure. (A) H&E-Stained Images of Tumors From the Four Experimental Groups in the Red-PDT Study: Untreated Control, Photosensitizer (PS) Alone, Light Exposure Alone, and Red-PDT. A Magnified View of the Boxed Region From the Red-PDT Group Is Shown in the Upper-Right Panel. (B) Corresponding H&E-Stained Images From the NIR-PDT Study, Including the Same Four Experimental Conditions, With the Magnified Image of the NIR-PDT Group Displayed in the Lower-Right Panel. No Conspicuous Tissue Damage Was Observed in the Control Groups (No Treatment, PS Alone, or Laser Light Alone) in Either Study. In Contrast, the PDT-Treated Groups Showed Marked Histological Evidence of Cell Death. In the Magnified Images, Specific Features of Necrotic and Apoptotic Cells Were Indicated With Arrowheads: Nuclear Chromatin Condensation (Pyknosis, Green), Nuclear Fragmentation (Karyorrhexis, Yellow), Nuclear Fading Due to DNA Degradation (Karyolysis, Red), and Anuclear Necrotic Cells With a Glossy, Homogeneous Appearance (Black). (Scale Bar = 50 μm; 20× Magnification for Left Eight Images, and Scale Bar = 50 μm; 40× Magnification for Right Two Images).

Confirming and Measuring Tumor Necrotic Depth by Colocalization of Fluorescence Imaging and Histology of Tumor Sections

To evaluate the depth of tumor necrosis induced by PDT, tumors were excised and vertically bisected through the center of the light-exposed region (Figure 8A). Based on previous findings by Jin et al, pHLIP accumulation correlates with active necrosis and co-localizes with necrotic cells undergoing pyknosis, karyorrhexis, and karyolysis, particularly at the periphery of necrotic zones. ²⁷ Therefore, the accumulation margin of AF546-pHLIP was used as a proxy for the boundary of necrosis. The distance from the tumor surface to the pHLIP accumulation margin was measured and used as an indicator of necrotic depth. Representative whole-tumor section images showing AF546-pHLIP fluorescence and corresponding H&E staining for tumors treated with Red-PDT and NIR-PDT were presented in Figures 8B and 8C, respectively. Merged images of AF546-pHLIP and H&E staining further confirmed the spatial correlation between fluorescence signal and histological necrosis. The measured necrotic depths induced by Red-PDT and NIR-PDT were compared and presented in a bar graph (Figure 8D).

Figure 8.

Confirmation and Measurement of Tumor Necrosis Depth Induced by Photodynamic Therapy (PDT). (A) Schematic Illustration of Tumor Sectioning Method Used to Assess the Depth of Necrosis Following PDT. (B) Representative Images of a Whole Tumor Section From the Red-PDT Group, Showing Accumulation of the Fluorescence From the Necrosis Marker AF546-pHLIP, Hematoxylin and Eosin (H&E) Staining of the Same Section, and a Merged Image of Both. A Magnified View of the Colocalized Necrotic Region Is Also Provided. (C) Representative Images of a Whole Tumor Section From the NIR-PDT Group, Showing AF546-pHLIP Fluorescence, H&E Staining, and the Merged Image, With a Magnified View of the Colocalized Necrotic Region. AF546-pHLIP Accumulates in Necrotic Regions, and Co-Localization With Histological Staining Confirms Necrosis Distribution and Depth. (Scale Bar = 1000 μm for Whole-Tumor Section Images.) (D) Quantitative Comparison of Necrosis Depth Induced by Red-PDT and NIR-PDT, Shown as a Bar Graph. Depth Was Measured From the Tumor Surface to the Deepest Boundary of AF546-pHLIP Accumulation. Statistical Analysis Was Performed Using Student's t-Test. Data Are Presented as Mean ± SD (n = 3 Tumors per Group).

Discussion

Previous studies have suggested that both red and NIR-activated PSs are promising for oncologic PDT.^11–13 Some reports have indicated that NIR light-triggered PDT possessing deeper penetration to lesion area and lower photodamage to normal tissue holds great potential for deep-seated tumor in vivo.^28–30 However, a fair and accurate comparison, along with an unbiased interpretation of their efficacy in vitro and in vivo settings, has not yet been conducted. To address this, we designed a controlled experiment using the same cell line/tumor model expressing the same target molecule. The PSs were delivered via the identical carrier system, and irradiation was performed using the same light dose and beam diameter (Figure 1). Prior to conducting this study, we speculated that NIR-PDT would exhibit superior phototherapeutic efficacy compared to Red-PDT due to its relatively deeper tissue penetration associated with longer wavelengths. However, our results from both in vitro and in vivo experiments under matched conditions revealed no significant difference in therapeutic efficacy between Red-PDT and NIR-PDT. One possible explanation is that the difference in wavelength between red and NIR light is relatively small, resulting in only a modest difference in photon energy, approximately 1.77 eV for 700 nm light versus 1.55 eV for 800 nm light. This energy difference may be insufficient to produce a meaningful difference in phototherapeutic outcomes under the tested conditions.

There are several reports in the literature comparing the phototherapeutic effects of blue and red light. For example, Helander et al reported that blue light exhibited several times greater phototoxic effects than red light in cancer cell lines treated with hexyl 5-aminolevulinate (HAL)-induced protoporphyrin IX (PpIX), which has a strong absorption peak at approximately 410 nm and a secondary peak around 630 nm. In their study, monolayer cells were incubated with HAL and then irradiated, so light scattering, penetration depth, and cell density did not have a significant impact on light transmission. ³¹ Meanwhile, Maytin et al reported that blue and red light were similarly effective in treating basal cell carcinoma in patients following the application of 5-aminolevulinic acid (ALA). ³² In a similar vein, it remains unclear whether there are meaningful differences in cancer therapeutic outcomes between Red-PDT and NIR-PDT, as this comparison has not been thoroughly investigated. To address this, we considered that different cell lines/tumor models may express varying levels of target molecules, potentially introducing bias in PS binding and therapeutic efficacy evaluation. Therefore, to ensure a fair comparison, we used the same cell line/tumor model, standardized the light dose and treatment schedule, and selected appropriate PSs and light sources with wavelengths matched to each PS.

This study was designed to address both challenges and uncertainties in comparing red and NIR-PDT. Active targeting strategies using specific molecules, such as antibodies against tumor-associated antigens, have been widely employed to deliver photosensitizing agents to neoplastic cells.^14,33,34 In the present study, we used the human monoclonal antibody trastuzumab (anti-HER-2) as a carrier to deliver the Tra-HLF647 (Red-PS) and Tra-ICG (NIR-PS) to A4 tumors overexpressing HER-2. This approach resolved the uncertainty regarding the feasibility of a direct comparison between Red-PDT and NIR-PDT by focusing on the same target. In this study, the commercially available, water-soluble dyes HLF647 and ICG were conjugated to Trastuzumab. ICG, a cyanine dye with peak absorption around 800 nm, has been approved by the US Food and Drug Administration (FDA) as a NIR clinical imaging agent. ¹⁶ Its application as a photosensitizer has also been reported to suppress tumor growth in xenograft models.^20,21,23 In contrast, HLF647 has primarily been used for immunostaining, with no previous reports on its photosensitizing potential. In our study, HLF647 exhibited no observable dark toxicity while demonstrating a therapeutic effect in both in vitro and in vivo experiments, suggesting its potential utility as a new photosensitizer. Furthermore, the absorbance spectra of both dyes closely matched the wavelengths of the designated light sources, making them suitable representatives of Red-PS and NIR-PS, respectively.

The accumulation of PSs at the target site was confirmed through both in vitro and in vivo experiments. Following overnight incubation of A4 cells with Tra-HLF647 or Tra-ICG, and after intravenous injection of tumor-bearing mice with the same conjugates, strong FI was observed not only in A4 cells (Figure 2C) but also in in vivo (Figures 5A, 5D) and ex vivo tumors (Figures 5C, 5F). These results indicate the specific and preferential accumulation of PSs in HER2-expressing cells/tumors. The membranous and subcellular localization of PSs, along with their activation within tumor tissues, lead to the generation of ROS, which can directly induce cytotoxicity in malignant tumor cells. ⁴ Serial in vivo fluorescence imaging revealed that PS accumulation in tumors peaked at 24–48 h after injection. A subsequent decrease in FI following PDT was observed, likely due to partial photobleaching of the PSs and the destruction of cancer cells. These findings provide evidence of successful light activation and therapeutic engagement.

To comprehensively evaluate and compare the potential effects of Tra-HLF647-mediated Red-PDT and Tra-ICG-mediated NIR-PDT, we conducted a series of in vitro and in vivo assessments, including cell viability imaging assay, intracellular ROS measurement, longitudinal monitoring of tumor volume changes, histological and IHC analyses of tumor sections, and measurements tumor necrotic depth. In cell viability imaging assays using fluorescence microscopy, both Red-PDT and NIR-PDT induced a similar degree of rapid cell death, indicating comparable phototoxicity (Figures 3A, 3B). This suggests that both PDTs were equally effective, highly phototoxic, and exhibited no significant dark toxicity to cells. Additionally, visualization and quantification of ROS generation in cell samples treated with Red-PDT and NIR-PDT revealed nearly identical results (Figures 4A, 4B, 4C). The ROS quantification experiment was performed once; therefore, although the observed trends suggest comparable ROS induction by Red-PDT and NIR-PDT, additional biological replicates are required to confirm statistical significance. Since these in vitro experiments were conducted on monolayer cells, light penetration was not a confounding factor.

The in vivo PDT regimen involved a single intravenous injection of the photosensitizer (Tra-HLF647 or Tra-ICG), followed by laser light irradiation (63.7 J/cm²) for two consecutive days (Figure 6A). Both Red-PDT and NIR-PDT significantly delayed tumor growth compared to other control groups. However, no significant difference in the relative tumor volume was observed between the two PDT groups (Figures 6B, 6C). Moreover, H&E-stained tumors sections from the respective PDT-treated groups revealed characteristic features of necrosis, including pyknosis, karyorrhexis, karyolysis, and anuclear necrotic cells (Figures 7A, 7B). In contrast, control groups treated with PS alone, light exposure alone, or no treatment showed no conspicuous tissue damage. Furthermore, whole-tumor H&E staining demonstrated distinct histological differences between irradiated and non-irradiated regions, confirming PDT-induced necrosis (Figures 8B, 8C).

We also evaluated laser light penetration by assessing the depth of necrosis. The distance from the illuminated tumor surface to the boundary of fluorescent necrosis marker (AF546-pHLIP) accumulation was used to estimate necrosis depth, as the accumulation of this marker coincided with active necrosis, particularly at the periphery of necrotic regions. ²⁷ This depth served as an indirect indicator to approximate laser light penetration, based on the assumption that the depth of marker accumulation correlates with the extent of light propagation within the tissue. Our results showed that the necrosis depth induced by both Red-PDT and NIR-PDT was slightly greater than 1000 μm, with no significant difference between the two groups. This finding suggests that red and NIR laser light penetration in our tumor model was nearly identical. However, the observed penetration depths were shallower than those reported in other review studies. For instance, red light penetration into skin is 4–5 mm, ³⁵ and penetration into brain tissue is even greater than in the skin. ³⁶ The optical properties of the tumor and other tissues—such as absorption, scattering, and reflection—are thought to be contributing factors to this discrepancy.

While this study provides valuable insights into the comparative efficacy of Red-PDT and NIR-PDT using representative PSs, several limitations should be acknowledged. First, the experiments were conducted using a single cell line/tumor model (A4) in immunodeficient mice, and therefore the results may not be fully generalizable to other tumor types with distinct biological characteristics, such as variations in vascularity, stromal composition, and immune microenvironment. Second, only two PSs representing red and near-infrared spectrum were evaluated. Although care was taken to select relevant PSs, the findings do not capture the full chemical diversity found in clinical and experimental PSs. Third, although we used a fluorescence-based necrosis marker to estimate the effective depth of light penetration, this method remains an indirect surrogate and does not fully account for optical heterogeneity or tissue scattering properties in vivo. Additionally, translation to human tumors may require caution, as mouse models often differ from human cancers in size, tissue depth, microenvironmental complexity, and pharmacokinetics. Therefore, stronger conclusions could be drawn by expanding the study to include multiple Red-PS and NIR-PS candidates, as well as a broader range of cell lines and tumor models, thereby allowing for more comprehensive data accumulation. Finally, although this study primarily focused on direct tumor necrosis ³⁷ as the principal mechanism of PDT-induced tumor destruction, other indirect mechanisms such as immune activation, vascular damage, and hyperthermal effects,^38,39 were not assessed. These pathways may contribute differently to the therapeutic outcomes of Red-PDT and NIR-PDT depending on PS properties and thus warrant further investigation in future studies.

Previous studies have explored the efficacy of PDT using either red or NIR wavelengths, often focusing on individual PSs; our study provided a side-by-side comparison under controlled conditions using standardized imaging and analysis. The overall findings of this study indicated that target-specific Red-PT and NIR-PT, utilizing their respective Red- and NIR-PSs, exhibit equivalent therapeutic efficacy against tumors in a preclinical tumor model. Our study challenges the assumption that NIR-PDT inherently outperforms Red-PDT and highlights the importance of considering PS characteristics when selecting treatment parameters. This contributes novel insights into the practical flexibility of PS wavelength selection in oncologic PDT and may inform rational design of future PSs, including dual-mode or broadband-activatable agents.

Conclusion

This study demonstrated that target-specific Red-PDT and NIR-PDT, when applied with their respective photosensitizers, achieve comparable therapeutic efficacy in a preclinical tumor model. These findings suggest that, under controlled experimental conditions, wavelength differences may not critically impact treatment outcomes, offering flexibility in fluorophore selection for future PS conjugate design. However, given the study's limited scope—using only two PSs and a single tumor model—further research is warranted to determine the generalizability of these results across diverse tumor types. Importantly, when target-specific delivery systems are appropriately employed, the high tumor accumulation of both Red and NIR-PSs can be harnessed for a range of clinical applications, including tumor detection, tumor screening, image-guided cancer detection, treatment planning, and protocol optimization, therapeutic outcome prediction, and minimizing the risk of late-stage clinical trial failure.

Supplemental Material

sj-docx-1-tct-10.1177_15330338251390292 - Supplemental material for Comparative Evaluation of Wavelength-Dependent Photodynamic Therapy Efficacy Using Representative Red and Near-Infrared Photosensitizers in a Single Tumor Model

Supplemental material, sj-docx-1-tct-10.1177_15330338251390292 for Comparative Evaluation of Wavelength-Dependent Photodynamic Therapy Efficacy Using Representative Red and Near-Infrared Photosensitizers in a Single Tumor Model by Winn Aung, Atsushi B Tsuji, Zhao-Hui Jin, Aya Sugyo, Chie Kajiwara and Tatsuya Higashi in Technology in Cancer Research & Treatment