Herein, we describe a synthetic route toward privileged 4-indolyl-3,5-diaryl-3-pyrrolin-2-ones based on a previously reported Friedel-Crafts reaction of 5-hydroxy-3,5-diaryl-3-pyrrolin-2-ones with indole. The intermediate 5-indolyl-3,5-diaryl-3-pyrrolin-2-ones are not isolated but further reacted under one-pot conditions, leading to the indole moiety migration from position C5 to position C4 of the 3-pyrrolin-2-one ring. The optimal reaction conditions found involved stirring with 2 equiv of aluminum chloride in 1,4-dioxane to complete the Friedel-Crafts step and then heating at 130 °C for 40 min in a microwave reactor to achieve the rearrangement step. Using the developed chemistry, a variety of compounds were prepared for biological testing, including those oxidized at C5 because 5-hydroxy-3-pyrrolin-2-ones have been reported to possess diverse biological properties as well. The synthesized compounds were tested for antiproliferative activities against MDA-MB-231 triple-negative breast cancer cells under normoxic and hypoxic conditions at a single concentration of 10 μM, and a number of compounds belonging to each of the series were identified to have noteworthy antiproliferative action under both normoxia and hypoxia. Several compounds from each series were further tested against ovarian cancer cells, and compounds from each series were capable of reducing cell viability of chemotherapy-resistant OVCAR-5 cells by as much as 75-80% at a concentration of 5 μM.

Pancreatic ductal adenocarcinoma (PDAC) remains highly resistant to chemotherapy and immunotherapy due to a dense tumor stroma and an immunosuppressive tumor microenvironment. In this study, we explored whether the photodynamic priming (PDP) effect, a fallout of Photodynamic therapy (PDT), an approved treatment, could improve the treatment responses in 3D mouse-derived organoids. The organoids reproduced key features of pancreatic tumors and showed strong resistance to chemotherapy (nanoliposomal irinotecan, nal-IRI) and immune checkpoint inhibitor (anti-PD-1) when used alone. PDP overcame this resistance by enhancing the uptake and activation of nal-IRI, thereby increasing tumor cell death. PDP also triggered immunogenic cell death, marked by the release of danger signals that promote immune activation. When organoids were co-cultured with autologous peripheral blood mononuclear cells (PBMCs), PDP enhanced immune-mediated tumor killing. Notably, combining PDP with low doses of chemotherapy and PD-1 blockade resulted in complete tumor eradication. These effects were associated with increased immune activation and improved responsiveness to immunotherapy.Together, these findings show that PDP remodels the pancreatic tumor microenvironment, enhances chemotherapy efficacy, and sensitizes tumors to immune checkpoint inhibitors. This strategy uses clinically approved agents and offers a promising, translatable approach to overcoming treatment resistance in pancreatic cancer.

Indocyanine green (ICG) is a clinically approved near-infrared fluorescent dye used in medical imaging and diagnostics but has limitations owing to its poor photostability in aqueous environments. This paper has explored the role of human serum albumin (HSA) and cholesterol in protecting ICG photostability. Pure ICG, an HSA-ICG complex, and an ICG–cholesterol colloidal assembly solution were stirred in the dark (control) and under continuous broadband irradiation (400–1600 nm, approximately 1.4 W), and absorption spectra (550–950 nm) were taken every 1 min, over 15 min. All the formulations were stable in the dark, with minimal total variance in absorbance. Pure ICG significantly photodegraded under irradiation (55 ± 2.75–65 ± 3.25% loss of maximum absorbance). The introduction of HSA and cholesterol limited the photodegradation, resulting in 15 ± 0.75–30 ± 1.5% and 25 ± 1.25% losses in maximum absorbance, respectively, upon irradiation. The modulators produced a significant increment in initial NIR absorbance (p < 0.001) and retained significantly high stability during irradiation (p < 0.01). Moreover, both modulators reduced photooxidative damage, as shown by the lower level of singlet oxygen (1O2) generation in the presence of HSA-ICG (35 ± 1.75%) and ICG–cholesterol (19 ± 0.95%) compared to pure ICG (57 ± 2.85% after 15 min). These results reveal that cholesterol is the best stabilizer of ICG photostability. By safely dissipating excitation energy via non-radiative decay, cholesterol demonstrates strong potential for enhancing ICG performance in photothermal therapy (PTT), whereas HSA remains the optimal modulator for near-infrared fluorescence imaging and photodynamic therapy.

Molecular fluorescence imaging is effective for tumor diagnosis but limited by low-depth profiling, which can be addressed by photoacoustic (PA) imaging. However, PA imaging has low sensitivity due to microenvironment-induced effects on exogenous contrast agents. Accordingly, the temporal and biophysical determinants of PA contrast in Cetuximab-IRDye800 conjugates should be performed to complement fluorescence-based diagnostics. In this study, we compare the temporal dynamics of PA and fluorescence signals from a Cetuximab-IRDye800 conjugate in a tumor xenograft model. We demonstrate that while fluorescence signal increases steadily over time after administration of Cetuximab-IRDye800, PA signal peaks early (~75% higher at 3 hours), followed by a decrease (~24% higher at 24 hours). Mechanistic analysis revealed formation of H-aggregates with Cetuximab-IRDye800 conjugation, which results in enhanced PA contrast, while receptor-mediated endocytosis disrupts these aggregates, reducing PA signal intensity over time. These findings underscore the complementary nature of PA and fluorescence imaging and emphasize timing as a critical factor for capturing peak PA contrast for tumor diagnostics.

Significance:Photodynamic therapy (PDT) for the treatment of oral cancers and oral potentially malignant lesions can be enhanced by the capability of the photosensitizer to serve as a fluorescence contrast agent for treatment guidance. The development of image-based dosimetry reporters can inform treatment progress in real time to avoid under-treatment, leading to incomplete response and recurrence. Aim:We investigate the hypothesis that imaging of protoporphyrin IX (PpIX) photoproduct (PP) accumulation may be leveraged as an implicit PDT dosimetry reporter for PDT using 5-aminolevulinic acid (ALA)-induced PpIX photosensitization. Approach:In initial spectroscopy studies, we investigate dose-dependent changes in absorption and fluorescence spectra of PpIX corresponding to PP accumulation during red light (635 nm) delivery. We use spectral analysis to select fluorescence excitation and spectral filtering components for PP imaging during treatment. We evaluate the capability for imaging PP accumulation concomitant with PpIX photobleaching in tissue phantoms, 3D oral squamous cell carcinoma (OSCC) models, and in murine xenografts. Results:Spectroscopy shows fluence-dependent changes in PpIX optical properties, and that excitation of photobleached PpIX with 450 nm light produces fluorescence emission associated with PpIX PPs. An existing handheld intraoral probe is shown to be capable of imaging dose-dependent PP accumulation with the addition of a spectral filter to isolate fluorescence emission longer than 650 nm. PP signal increases concomitant with PpIX photobleaching in a fluence-dependent manner and correlates with the extent of cytotoxic response in 3D cultures. PP accumulation is also observed to occur concomitantly with photobleaching in OSCC subcutaneous xenografts. Conclusions:Overall, the results show that imaging of PP accumulation is feasible by adapting traditional photodiagnosis optical components and may serve as a dosimetry reporter for ALA-PDT, which is complementary to the measurement of PpIX photobleaching.

Cervical cancer represents a significant global health challenge. Photodynamic therapy (PDT) appears to be a promising, minimally invasive alternative to standard treatments. However, the clinical efficacy of PDT is sometimes limited by the low solubility and aggregation of photosensitizers, their non-selective distribution in the body, hypoxia in the tumor microenvironment, and limited light penetration. Recent advances in nanoparticle and nanocomposite platforms have addressed these challenges by integrating multiple functional components into a single delivery system. By encapsulating or conjugating photosensitizers in biodegradable matrices, such as mesoporous silica, organometallic structures and core–shell construct nanocarriers increase stability in water and extend circulation time, enabling both passive and active targeting through ligand decoration. Up-conversion and dual-wavelength responsive cores facilitate deep light conversion in tissues, while simultaneous delivery of hypoxia-modulating agents alleviates oxygen deprivation to sustain reactive oxygen species generation. Controllable “motor-cargo” constructs and surface modifications improve intratumoral diffusion, while aggregation-induced emission dyes and plasmonic elements support real-time imaging and quantitative monitoring of therapeutic response. Together, these multifunctional nanosystems have demonstrated potent cytotoxicity in vitro and significant tumor suppression in vivo in mouse models of cervical cancer. Combining targeted delivery, controlled release, hypoxia mitigation, and image guidance, engineered nanoparticles provide a versatile and powerful platform to overcome the current limitations of PDT and pave the way toward more effective, patient-specific treatments for cervical malignancies. Our review of the literature summarizes studies on nanoparticles and nanocomposites used in PDT monotherapy for cervical cancer, published between 2023 and July 2025.

Photodynamic therapy (PDT) is a promising, minimally invasive treatment for cervical cancer, but its efficacy is significantly limited by hypoxia—oxygen deficiency in the tumour microenvironment. The aim of this study was to present strategies to counteract hypoxia in PDT using the latest nanotechnologies. Based on a review of the literature available in PubMed/MEDLINE, Scopus, and Web of Science databases, covering the period from January 2024 to March 2025, nine original in vivo studies were identified that investigated the use of nanoparticle-based strategies to overcome hypoxia and enhance the efficacy of PDT in cervical cancer. A variety of approaches to improve tumour oxygenation are described, including the catalytic decomposition of hydrogen peroxide (H2O2) with manganese oxide (MnO2), the use of bimetallic nanozymes (e.g., Au2Pt), and FeOOH structures and oxygen storage and control systems (e.g., endoperoxides). Strategies to reduce oxygen consumption by cancer cells, such as nitric oxide (NO) release or inhibition of mitochondrial oxidative phosphorylation, are also discussed. The review shows that appropriately designed nanoparticles can effectively counteract hypoxia, enhancing the efficacy of PDT by intensifying reactive oxygen species (ROS) generation and modulating HIF-1α factor expression. The strategies presented here have the potential to significantly improve the efficacy of photodynamic therapy in the treatment of cervical cancer, especially under conditions of limited oxygen availability.

The poor response of pancreatic ductal adenocarcinoma (PDAC) to treatment, including immunotherapy, is attributed to its tumor microenvironment (TME). An ongoing challenge is the desmoplastic and immunosuppressed TME that evades immune surveillance. Here, we investigate transient modulation of the TME to overcome immunosuppression using a light-activated process, termed photodynamic priming (PDP). As a first step, this study captures the temporal dynamics of variations in immune infiltrates and subsequent immune responses in the TME, spleen, and blood of the KPC mouse model of PDAC post-PDP. In response to PDP, there were transient increases in tumor infiltrating lymphocytes (TIL) in tumors. The TIL population post-PDP includes an enrichment of CD8+ T cells, accompanied by temporal increases in PD-1, CTLA-4, and TIM-3 immune checkpoints on both CD8+ T and CD4+ T cells. Significant increases in CD11C+MHC-11+ dendritic cells and proliferating lymphocytes are observed in the spleen within several hours post-tumor PDP, suggesting initiation of adaptive immune responses. These observations are followed by an expansion of CD44+CD62-CD8+ effector memory T cells in the blood over several days as evidence of a systemic immune response. Post-PDP TME alterations also included the reduced formation of blood (CD31+) and lymphatic (Lyve-1+) vessels as well as decreases in PD-L1 and collagen content. Collectively, these data suggest that PDP helps to mitigate immunosuppressive mechanisms and promote enhanced tumor permeability. The temporal dynamics of the processes elucidated here pave the way to develop strategies in future work for combined PDP-immunotherapy utilizing the immune checkpoint expression dynamics for precision therapy.

Photoimmunotherapy (PIT) involves the targeted delivery of a photosensitizer through antibody conjugation, which, upon binding to its cellular target and activation by external irradiation, induces localized toxicity. This approach addresses several limitations of conventional cancer therapies, such as chemo- and radiotherapies, which result in off-target effects that significantly reduce patient quality of life. Furthermore, PIT improves on the challenges encountered with photodynamic therapy (PDT), such as nonspecific localization of the photosensitizer, which often results in unintended toxicities. Although PIT was first proposed in the early 1980s, its clinical applications have been constrained by limitations in antibody engineering, conjugation chemistries, and optical technologies. However, recent advances in antibody-drug conjugate (ADC) research and the emergence of sophisticated laser technologies have greatly benefited the broader applicability of PIT. Notably, the first near-infrared photoimmunotherapy (NIR-PIT) treatment for head and neck cancer has been approved in Japan and is currently in phase III clinical trials in the USA. A significant advantage of PIT over traditional ADCs in cancer management is the agnostic nature of PDT, making it more adaptable to different tumor types. Specifically, PIT can act on cancer stem cells and cancer cells displaying treatment resistance and aggressive phenotypes-a capability beyond the scope of ADCs alone. This review provides an overview of the mechanism of action of NIR-PIT, highlighting its adaptability and application in cancer therapeutics, and concludes by exploring the potential of PIT in advancing cancer treatments.

Molecular fluorescence-guided surgery has shown promise for tumor margin delineation but is limited by its depth profiling capability. Interestingly, most fluorophores, either clinically approved or in clinical trials, can also be used as photoacoustic contrast agents, yet their use is limited due to the low light fluence permitted for clinical use and the limited sensitivity of current photoacoustic imaging systems. There is therefore an urgent unmet need to establish methods for enhancing contrast in molecular targeted PA imaging which could potentially complement and overcome limitations in molecular fluorescence guided therapies. In this study, we compare the photoacoustic (PA) and fluorescence imaging capabilities of a cetuximab-IRDye800 conjugate in a subcutaneous tumor xenograft model. We demonstrate that while the fluorescence signal increases steadily over time after administration of cetuximab-IRDye800, PA signal peaks early (~2 fold higher at 6-hour as compared to pre-injection controls) and then decreases (~1.3 fold higher at 24-hour as compared to pre-injection controls). This pattern aligns with previous findings using other antibody-conjugated PA contrast agents. Mechanistically, we demonstrate that the formation of H-aggregates upon antibody conjugation enhances PA contrast of the IRDye800. The disruption of these H-aggregates, as the antibody-dye conjugate is degraded post receptor-mediated endocytosis, decreases PA signal intensity. The timeframe of maximum PA signal and decrease thereafter is consistent with the time frame of receptor-mediated endocytosis of cetuximab-IRDye800. Our data suggests that tumor cell surface binding results in peak PA signal while lysosomal localization and degradation results in a significant drop in PA signal. Our study sheds light on the distinct temporal dynamics of PA and fluorescence signals of Cetuximab-IRDye800 conjugate and we propose that optimizing IRDye800 conjugation to antibodies can further enhance PA signal intensity when timed to precisely to capture IRDye800 in an H-aggregate form.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia which is considered to be a primary cause of treatment resistance observed in these tumors. Desmoplasia also plays a major role in mediating an immunosuppressive microenvironment and restricting immune cell infiltration in PDAC. In this study, using a syngeneic orthotopic immunocompetent KPC PDAC model, we demonstrate that PDT using Visudyne® can alter the tumor microenvironment, enhances immune cell infiltration, significantly reducing tumor growth and increasing survival in combination with anti-PD1.

Abstract The clinical uses of anthracyclines (e.g., doxorubicin) are hampered especially by dose-dependent cardiac toxicity, resulting in a narrow therapeutic window. Doxorubicin formulations have marginally improved its therapeutic efficacy while suffering from difficult and costly manufacturing, poor characterization and quality control, limited scalability in production, and disappointingly low drug loading. This study employs DNA fragments extracted from the salmon sperm cells, which is known for high biocompatibility in various biomedical application, to self-assemble via intercalation by doxorubicin. DNA/DOX nanocomplexes are easy in manufacturing, high scalability, straightforward characterization and quantification, and efficiency in drug loading. Doxorubicin and DNA fragments were dissolved in water and mixed at the optimized 1:6 weight ratio, followed by stirring and incubation for 30 min. DNA/DOX nanocomplexes were characterized for doxorubicin loading and release, size, surface charge, and morphology by UV/fluorescence spectroscopy, stable isotope tracer ultrafiltration assay (SITUA), dynamic light scattering particle analysis, and transmission electron microscopy. The anticancer efficacy of the DNA/DOX nanocomplexes were tested in vitro and in vivo using syngeneic and PDX tumor models. Along with PD/PK studies, DNA/DOX nanocomplexes were also evaluated for their systemic and cardiac toxicity. Characterization and quantification studies revealed DNA/DOX nanocomplexes to be relatively monodisperse with a size of 40 nm in diameter, efficient for drug encapsulation (~100%) and drug loading (14.3%, w/w), and stable for storage while readily releasing doxorubicin in a cell. The self-assembled nanocomplexes were formed when doxorubicin inter- and intra-molecularly chelated DNA fragments. DNA/DOX nanocomplexes were more efficient in killing cancer cells in vitro than both doxorubicin and the liposomal doxorubicin Doxil. PD/PK studies demonstrated that DNA/DOX nanocomplexes circulated longer with less accumulation in the heart than doxorubicin, which altogether lower toxicity. A repeated administration of the DNA/DOX nanocomplexes to Sprague-Dawley rats exhibited a substantially lower cardiotoxicity score than that of doxorubicin. The improved anti-tumor efficacy of the DNA/DOX nanocomplexes were finally confirmed using a syngeneic lymphoma mouse model and PDX mouse models of triple-negative breast and ovarian cancers. Complexing doxorubicin with DNA fragments addresses the current technological challenges in achieving an efficient and safe therapy for a broad range of cancers. DNA/DOX nanocomplexes are amenable to rigorous chemistry, manufacturing, and controls (CMC) evaluation with proven anti-cancer efficacy and improved safety profiles. Clinical trials on DNA/DOX nanocomplexes for cancer chemotherapy are warranted to fully validate their clinical utilities. Citation Format: Minhyeong Park, Youngwoo Kim, Hyunchu Cho, Saad Mohammad, Yeon Su Choi, Hyunjoo Lee, Seonah Lee, Edward Cedrone, Barry Neun, Maria Dobrovolskaia, David Fruman, Juwan Kim, Young Jik Kwon. Efficient and safe delivery of doxorubicin by DNA fragments: A full preclinical study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5735.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has been shown to selectively destroy tumor in a metastatic mouse model. However, the photoimmunoconjugate (PIC) used for taPIT includes a small fraction of non-covalently associated (free) benzoporphyrin derivative (BPD), which leads to non-specific killing in vitro. Here, we report a new treatment protocol for patient-derived primary tumor cell cultures ultrasensitive to BPD photodynamic therapy (BPD-PDT). Based on free BPD efflux dynamics, the updated in vitro taPIT protocol precludes non-specific BPD-PDT by silencing the effect of free BPD. Following incubation with PIC, incubating cells with PIC-free medium allows time for expulsion of free BPD whereas BPD covalently bound to PIC fragments is retained. Administration of the light dose after the intracellular free BPD drops below the threshold for inducing cell death helps to mitigate non-specific damage. In this study, we tested two primary ovarian tumor cell lines that are intrinsically chemoresistant, yet ultrasensitive to BPD-PDT such that small amounts of free BPD (a few percent of the total BPD dose) lead to potent induction of cell death upon irradiation. The modifications in the protocol suggested here improve in vitro taPIT experiments that lack in vivo mechanisms of free BPD clearance (i.e., lymph and blood flow). Using patient-derived primary cancer cell models and conventional cell lines with varying levels of epidermal growth factor receptor (EGFR) expression, the protocol for performing tumor-targeted, activatable photoimmunotherapy (taPIT) has been refined to avoid non-specific cell death in vitro. This protocol exploits the slow uptake and long retention of proteolyzed photoimmunoconjugate (PIC) fragments versus the relatively fast free photosensitizer efflux dynamics in cells.image

SignificanceDelineating tumor margins during surgery and eliminating residual disease is a major challenge in the treatment of oral cancers. The dual function antibody conjugate (DFAC) developed in this study can enable 3D-imaging and destroy residual microscopic disease by photoimmunotherapy in a single intra-operative setting.ApproachDFAC was synthesized by routine carbodiimide crosslinker chemistry. Multi-modal imaging (Photoacoustic and fluorescence imaging) and photoimmunotherapy was performed on oral cancer spheroids.ResultsPhotoacoustic imaging could detect tumor spheroids, even of the smallest size (0.01 mm3), while fluorescence imaging could detect only larger spheroids with volumes greater than 0.049 mm3 and treated with a higher DFAC concentration. Photoimmunotherapy showed a spheroid size dependent response, with smaller spheroids of volumes 0.018 mm3 and lower showing no recurrence.ConclusionsThe complementary imaging and treatment capabilities of DFAC could help in minimizing healthy tissue resection and enhance recurrence free survival in oral cancer patients.

Photoacoustic imaging using external contrast agents is emerging as a powerful modality for real-time molecular imaging of deep-seated tumors. There are several chromophores, such as indocyanine green and IRDye800, that can potentially be used for photoacoustic imaging; however, their use is limited due to several drawbacks, particularly photostability. There is, therefore, an urgent need to design agents to enhance contrast in photoacoustic imaging. Naphthalocyanine dyes have been demonstrated for their use as photoacoustic contrast agents; however, their low solubility in aqueous solvents and high aggregation propensity limit their application. In this study, we report the synthesis and characterization of silicon-centered naphthalocyanine dyes with high aqueous solubility and near infra-red (NIR) absorption in the range of 850–920 nm which make them ideal candidates for photoacoustic imaging. A series of Silicon-centered naphthalocyanine dyes were developed with varying axial and peripheral substitutions, all in an attempt to enhance their aqueous solubility and improve photophysical properties. We demonstrate that axial incorporation of charged ammonium mesylate group enhances water solubility. Moreover, the incorporation of peripheral 2-methoxyethoxy groups at the α-position modulates the electronic properties by altering the π-electron delocalization and enhancing photoacoustic signal amplitude. In addition, all the dyes were synthesized to incorporate an N-hydroxysuccinimidyl group to enable further bioconjugation. In summary, we report the synthesis of water-soluble silicon-centered naphthalocyanine dyes with a high photoacoustic signal amplitude that can potentially be used as contrast agents for molecular photoacoustic imaging.

Photodynamic therapy (PDT), a combination of light, molecular oxygen and a photosensitizing dye, has gained attention as a promising technique to treat various types of cancers. Among all the photosensitizers reported so far, ruthenium(II) polypyridyl complexes exhibit unique photophysical and photobiological features owing to their photostability, Its triplet excited states, and ability to undergo both 'type I' and 'type II' reactions in their photodynamic action. We report the synthesis of a novel Ru(II) complex containing one 2,2 '-biimidazole (bim) and two tetramethylphenanthroline (tmp) ligands that sensitizes the simultaneous production of superoxide anion (O-2(center dot-)) and singlet oxygen (O-1(2)) upon irradiation with blue-green light. To improve its solubility and bioavailability, a zero-order degradation-controlled release formulation based on self-assembled hyaluronic acid (HA)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) was prepared for its topical application in oral cancer cells (TR146 cell line). These NPs (152 nm diameter) showed 70 % Ru-complex encapsulation efficiency, high physiological stability, low polydispersity index (0.12), and a sensitizer release enhanced by the hyal-uronidase enzyme overexpressed in many cancer cells. Both the free complex and its nanocarrier are internalized by the TR146 cells, displaying > 90 % in vitro cytotoxicity under 470 nm activation (50 J cm(-2)), highlighting their potential as PDT agents. The Ru(II) complex loaded nanocarrier developed in this study can be potentially effective in the treatment of oral cancers.