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Glioblastoma stem cells (GSCs) are potent tumor initiators resistant to radiochemotherapy, and this subpopulation is hypothesized to re-populate the tumor milieu due to selection following conventional therapies. Here, we show that 5-aminolevulinic acid (ALA) treatment-a pro-fluorophore used for fluorescence-guided cancer surgery-leads to elevated levels of fluorophore conversion in patient-derived GSC cultures, and subsequent red light-activation induces apoptosis in both intrinsically temozolomide chemotherapy-sensitive and -resistant GSC phenotypes. Red light irradiation of ALA-treated cultures also exhibits the ability to target mesenchymal GSCs (Mes-GSCs) with induced temozolomide resistance. Furthermore, sub-lethal light doses restore Mes-GSC sensitivity to temozolomide, abrogating GSC-acquired chemoresistance. These results suggest that ALA is not only useful for fluorescence-guided glioblastoma tumor resection, but that it also facilitates a GSC drug-resistance agnostic, red light-activated modality to mop up the surgical margins and prime subsequent chemotherapy.
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A feature issue is being presented by a team of guest editors containing papers based on studies presented at the Optical Molecular Probes, Imaging and Drug Delivery conference as part of the Optica Biophotonics Congress in Vancouver, Canada from April 24-27, 2023.
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Hypoxia in solid tumors, including head and neck cancer (HNC), contributes to treatment resistance, aggressive phenotypes, and poor clinical outcomes. Perfluorocarbon nanodroplets have emerged as promising oxygen carriers to alleviate tumor hypoxia. However, a thorough characterization of the hypoxia alleviation effects in terms of sustenance of oxygenated environments have not been thoroughly studied. In this study, we developed and characterized perfluoropentane nanodroplets (PFP NDs) for co-delivery of oxygen and the photoactivatable drug or photosensitizer benzoporphyrin derivative (BPD) to hypoxic HNC spheroids. The PFP NDs exhibited excellent stability, efficient oxygen loading/release, and biocompatibility. Using 3D multicellular tumor spheroids of FaDu and SCC9 HNC cells, we demonstrated the ability of oxygenated PFP NDs to penetrate the hypoxic core and alleviate hypoxia, as evidenced by reduced fluorescence of a hypoxia-sensing reagent and downregulation of hypoxia-inducible factors HIF-1α and HIF-2α. BPD-loaded PFP NDs successfully delivered the photosensitizer into the spheroid core in a time-dependent manner. These findings highlight the potential of PFP NDs as a co-delivery platform to overcome hypoxia-mediated treatment resistance and improve therapy outcomes in HNC.
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Pancreatic cancer (PC) is a highly lethal malignancy and the third leading cause of cancer deaths in the U.S. Despite major innovations in imaging technologies, there are limited surrogate radiographic indicators to aid in therapy planning and monitoring. Amongst the various imaging techniques Ultrasound-guided photoacoustic imaging (US-PAI) is a promising modality based on endogenous blood (hemoglobin) and blood oxygen saturation (StO 2 ) contrast to monitor response to anti-angiogenic therapies. Adaptation of US-PAI to the clinical realm requires macroscopic configurations for adequate depth visualization, illuminating the need for surrogate radiographic markers, including the tumoral microvessel density (MVD). In this work, subcutaneous xenografts with PC cell lines AsPC-1 and MIA-PaCa-2 were used to investigate the effects of receptor tyrosine kinase inhibitor (sunitinib) treatment on MVD and StO 2 . Through histological correlation, we have shown that regions of high and low vascular density (HVD and LVD) can be identified through frequency domain filtering of macroscopic PA images which could not be garnered from purely global analysis. We utilized vascular regional analysis (VRA) of treatment-induced StO 2 and total hemoglobin (HbT) changes. VRA as a tool to monitor treatment response allowed us to identify potential timepoints of vascular remodeling, highlighting its ability to provide insights into the TME not only for sunitinib treatment but also other anti-angiogenic therapies.
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Tissue engineering is a dynamic field focusing on the creation of advanced scaffolds for tissue and organ regeneration. These scaffolds are customized to their specific applications and are often designed to be complex, large structures to mimic tissues and organs. This study addresses the critical challenge of effectively characterizing these thick, optically opaque scaffolds that traditional imaging methods fail to fully image due to their optical limitations. We introduce a novel multi-modal imaging approach combining ultrasound, photoacoustic, and acoustic radiation force impulse imaging. This combination leverages its acoustic-based detection to overcome the limitations posed by optical imaging techniques. Ultrasound imaging is employed to monitor the scaffold structure, photoacoustic imaging is employed to monitor cell proliferation, and acoustic radiation force impulse imaging is employed to evaluate the homogeneity of scaffold stiffness. We applied this integrated imaging system to analyze melanoma cell growth within silk fibroin protein scaffolds with varying pore sizes and therefore stiffness over different cell incubation periods. Among various materials, silk fibroin was chosen for its unique combination of features including biocompatibility, tunable mechanical properties, and structural porosity which supports extensive cell proliferation. The results provide a detailed mesoscale view of the scaffolds' internal structure, including cell penetration depth and biomechanical properties. Our findings demonstrate that the developed multimodal imaging technique offers comprehensive insights into the physical and biological dynamics of tissue-engineered scaffolds. As the field of tissue engineering continues to advance, the importance of non-ionizing and non-invasive imaging systems becomes increasingly evident, and by facilitating a deeper understanding and better characterization of scaffold architectures, such imaging systems are pivotal in driving the success of future tissue-engineering solutions.
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Photoacoustic (PA) imaging is hybrid imaging modality with good optical contrast and spatial resolution. Portable, cost-effective, smaller footprint light emitting diodes (LEDs) are rapidly becoming important PA optical sources. However, the key challenge faced by the LED-based systems is the low light fluence that is generally compensated by high frame averaging, consequently reducing acquisition frame-rate. In this study, we present a simple deep learning U-Net framework that enhances the signal-to-noise ratio (SNR) and contrast of PA image obtained by averaging low number of frames. The SNR increased by approximately four-fold for both in-class in vitro phantoms (4.39 ± 2.55) and out-of-class in vivo models (4.27 ± 0.87). We also demonstrate the noise invariancy of the network and discuss the downsides (blurry outcome and failure to reduce the salt & pepper noise). Overall, the developed U-Net framework can provide a real-time image enhancement platform for clinically translatable low-cost and low-energy light source-based PA imaging systems.
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Procesamiento de Imagen Asistido por Computador , Fantasmas de Imagen , Técnicas Fotoacústicas , Relación Señal-Ruido , Técnicas Fotoacústicas/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Factores de Tiempo , Animales , Ratones , Aprendizaje Profundo , LuzRESUMEN
Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) for head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual-function antibody conjugate (DFAC) for multimodal imaging and photoimmunotherapy (PIT) of EGFR-overexpressing cancer cells. The DFAC reported previously and used in the present study comprises an EGFR-targeted antibody, cetuximab, conjugated to benzoporphyrin derivative (BPD) for fluorescence imaging and PIT and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3) developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response, with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess the presence of tumor on a macroscopic scale, followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intraoperative setting.
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Neoplasias de Cabeza y Cuello , Inmunoconjugados , Técnicas Fotoacústicas , Humanos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Fototerapia/métodos , Inmunoterapia/métodos , Inmunoconjugados/uso terapéutico , Neoplasias de Cabeza y Cuello/terapia , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Receptores ErbB , Línea Celular Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Depth-dependent fluence-compensation in photoacoustic (PA) imaging is paramount for accurate quantification of chromophores from deep tissues. Here we present a user-friendly toolkit named PHANTOM (PHotoacoustic ANnotation TOolkit for MATLAB) that includes a graphical interface and assists in the segmentation of ultrasound-guided PA images. We modelled the light source configuration with Monte Carlo eXtreme and utilized 3D segmented tissues from ultrasound to generate fluence maps to depth compensate PA images. The methodology was used to analyze PA images of phantoms with varying blood oxygenation and results were validated with oxygen electrode measurements. Two preclinical models, a subcutaneous tumor and a calcified placenta, were imaged and fluence-compensated using the PHANTOM toolkit and the results were verified with immunohistochemistry. The PHANTOM toolkit provides scripts and auxiliary functions to enable biomedical researchers not specialized in optical imaging to apply fluence correction to PA images, enhancing accessibility of quantitative PAI for researchers in various fields.
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In the past decade, photoacoustic (PA) imaging has attracted a great deal of popularity as an emergent diagnostic technology owing to its successful demonstration in both preclinical and clinical arenas by various academic and industrial research groups. Such steady growth of PA imaging can mainly be attributed to its salient features, including being non-ionizing, cost-effective, easily deployable, and having sufficient axial, lateral, and temporal resolutions for resolving various tissue characteristics and assessing the therapeutic efficacy. In addition, PA imaging can easily be integrated with the ultrasound imaging systems, the combination of which confers the ability to co-register and cross-reference various features in the structural, functional, and molecular imaging regimes. PA imaging relies on either an endogenous source of contrast (e.g., hemoglobin) or those of an exogenous nature such as nano-sized tunable optical absorbers or dyes that may boost imaging contrast beyond that provided by the endogenous sources. In this review, we discuss the applications of PA imaging with endogenous contrast as they pertain to clinically relevant niches, including tissue characterization, cancer diagnostics/therapies (termed as theranostics), cardiovascular applications, and surgical applications. We believe that PA imaging's role as a facile indicator of several disease-relevant states will continue to expand and evolve as it is adopted by an increasing number of research laboratories and clinics worldwide.
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Significance: India has one of the highest rates of oral squamous cell carcinoma (OSCC) in the world, with an incidence of 15 per 100,000 and more than 70,000 deaths per year. The problem is exacerbated by a lack of medical infrastructure and routine screening, especially in rural areas. New technologies for oral cancer detection and timely treatment at the point of care are urgently needed. Aim: Our study aimed to use a hand-held smartphone-coupled intraoral imaging device, previously investigated for autofluorescence (auto-FL) diagnostics adapted here for treatment guidance and monitoring photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence (FL). Approach: A total of 12 patients with 14 buccal mucosal lesions having moderately/well-differentiated micro-invasive OSCC lesions (<2 cm diameter and <5 mm depth) were systemically (in oral solution) administered three doses of 20 mg/kg ALA (total 60 mg/kg). Lesion site PpIX and auto-FL were imaged using the multichannel FL and polarized white-light oral cancer imaging probe before/after ALA administration and after light delivery (fractionated, total 100 J/cm2 of 635 nm red LED light). Results: The handheld device was conducive for access to lesion site images in the oral cavity. Segmentation of ratiometric images in which PpIX FL is mapped relative to auto-FL enabled improved demarcation of lesion boundaries relative to PpIX alone. A relative FL (R-value) threshold of 1.4 was found to segment lesion site PpIX production among the patients with mild to severe dysplasia malignancy. The segmented lesion size is well correlated with ultrasound findings. Lesions for which R-value was >1.65 at the time of treatment were associated with successful outcomes. Conclusion: These results indicate the utility of a low-cost, handheld intraoral imaging probe for image-guided PDT and treatment monitoring while also laying the groundwork for an integrated approach, combining cancer screening and treatment with the same hardware.
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Carcinoma de Células Escamosas , Neoplasias de la Boca , Fotoquimioterapia , Humanos , Ácido Aminolevulínico/uso terapéutico , Teléfono Inteligente , Neoplasias de la Boca/patología , Fotoquimioterapia/métodos , Protoporfirinas/metabolismo , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
There is an increasing need for 3D ultrasound and photoacoustic (USPA) imaging technology for real-time monitoring of dynamic changes in vasculature or molecular markers in various malignancies. Current 3D USPA systems utilize expensive 3D transducer arrays, mechanical arms or limited-range linear stages to reconstruct the 3D volume of the object being imaged. In this study, we developed, characterized, and demonstrated an economical, portable, and clinically translatable handheld device for 3D USPA imaging. An off-the-shelf, low-cost visual odometry system (the Intel RealSense T265 camera equipped with simultaneous localization and mapping technology) to track free hand movements during imaging was attached to the USPA transducer. Specifically, we integrated the T265 camera into a commercially available USPA imaging probe to acquire 3D images and compared it to the reconstructed 3D volume acquired using a linear stage (ground truth). We were able to reliably detect 500 µm step sizes with 90.46% accuracy. Various users evaluated the potential of handheld scanning, and the volume calculated from the motion-compensated image was not significantly different from the ground truth. Overall, our results, for the first time, established the use of an off-the-shelf and low-cost visual odometry system for freehand 3D USPA imaging that can be seamlessly integrated into several photoacoustic imaging systems for various clinical applications.
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Several molecular-targeted imaging and therapeutic agents are in clinical trials for image-guided surgery and photoimmunotherapy (PIT) of head and neck cancers. In this context, we have previously reported the development, characterization, and specificity of a dual function antibody conjugate (DFAC) for multi-modal imaging and photoimmunotherapy (PIT) of EGFR over-expressing cancer cells. The DFAC reported previously and used in the present study, comprises of an EGFR targeted antibody - Cetuximab conjugated to Benzoporphyrin derivative (BPD) for fluorescence imaging and PIT, and a Si-centered naphthalocyanine dye for photoacoustic imaging. We report here the evaluation and performance of DFAC in detecting microscopic cancer spheroids by fluorescence and photoacoustic imaging along with their treatment by PIT. We demonstrate that while fluorescence imaging can detect spheroids with volumes greater than 0.049 mm3, photoacoustic imaging-based detection was possible even for the smallest spheroids (0.01 mm3), developed in the study. When subjected to PIT, the spheroids showed a dose-dependent response with smaller spheroids (0.01 and 0.018 mm3) showing a complete response with no recurrence when treated with 100 J/cm2. Together our results demonstrate the complementary imaging and treatment capacity of DFAC. This potentially enables fluorescence imaging to assess tumor presence on a macroscopic scale followed by photoacoustic imaging for delineating tumor margins guiding surgical resection and elimination of any residual microscopic disease by PIT, in a single intra-operative setting.
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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.
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Colorantes , Técnicas Fotoacústicas , Medios de Contraste , Solubilidad , Silicio , Imagen Molecular , Agua , Técnicas Fotoacústicas/métodos , Colorantes FluorescentesRESUMEN
Comprehensive characterization of biomedical imaging systems require phantoms that are easy to fabricate and can mimic human tissue. Additionally, with the arrival of engineered tissues, it is key to develop phantoms that can mimic bioengineered samples. In ultrasound and photoacoustic imaging, water-soluble phantom materials such as gelatin undergo rapid degradation while polymer-based materials such as polyvinyl alcohol are not conducive for generating bioengineered tissues that can incorporate cells. Here we propose silk protein-based hydrogels as an ultrasound and photoacoustic phantom material that has potential to provide a 3D environment for long-term sustainable cell growth. Common acoustic, optical, and biomechanical properties such as ultrasound attenuation, reduced scattering coefficient, and Young's modulus were measured. The results indicate that silk acoustically mimics many tissue types while exhibiting similar reduced optical scattering in the wavelength range of 400-1200 nm. Furthermore, silk-based materials can be stored long-term with no change in acoustic and optical properties, and hence can be utilized to assess the performance of ultrasound and photoacoustic systems.
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Cholesteatoma is a potentially serious complication of chronic ear infections and requires surgical intervention for definitive management. Long-term complications include a frequent need for repeat surgical intervention for disease recurrence, and techniques to improve efficacy of single-stage surgery are an important area of continued research. This study investigates a novel application of the photosensitizer immune conjugate (PIC) cetuximab-benzoporphyrin derivative (Cet-BPD) for in vitro localization of human cholesteatoma tissue, coupled with an in vivo safety study for middle ear application of Cet-BPD in a murine model. In fresh human cholesteatoma tissues, Cet-BPD demonstrates selective localization to the hyperplastic squamous cell tissue associated with cholesteatoma, without localizing to other tissues such as middle ear mucosa. Applied to the murine middle ear, Cet-BPD does not demonstrate any deleterious effect on murine hearing when assessed by any of auditory brainstem response (ABR) thresholds, distortion product otoacoustic emission thresholds, or ABR wave I amplitudes. These findings demonstrate the technical promise and encouraging safety profile for the use of PICs for intraoperative localization and treatment of cholesteatoma.
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Colesteatoma del Oído Medio , Humanos , Ratones , Animales , Colesteatoma del Oído Medio/diagnóstico por imagen , Colesteatoma del Oído Medio/cirugía , Oído Medio , Diagnóstico por Imagen , Colorantes , Pruebas AuditivasRESUMEN
Pancreatic cancer is a disease with an incredibly poor survival rate. As only about 20% of patients are eligible for surgical resection, neoadjuvant treatments that can relieve symptoms and shrink tumors for surgical resection become critical. Many forms of treatments rely on increased vulnerability of cancerous cells, but tumors or regions within the tumors that may be hypoxic could be drug resistant. Particularly for neoadjuvant therapies such as the tyrosine kinase inhibitors utilized to shrink tumors, it is critical to monitor changes in vascular function and hypoxia to predict treatment efficacy. Current clinical imaging modalities used to obtain structural and functional information regarding hypoxia or oxygen saturation (StO2) do not provide sufficient depth penetration or require the use of exogenous contrast agents. Recently, ultrasound-guided photoacoustic imaging (US-PAI) has garnered significant popularity, as it can noninvasively provide multiparametric information on tumor vasculature and function without the need for contrast agents. Here, we built upon existing literature on US-PAI and demonstrate the importance of changes in StO2 values to predict treatment response, particularly tumor growth rate, when the outcomes are suboptimal. Specifically, we image xenograft mouse models of pancreatic adenocarcinoma treated with suboptimal doses of a tyrosine kinase inhibitor cabozantinib. We utilize the US-PAI data to develop a multivariate regression model that demonstrates that a therapy-induced reduction in tumor growth rate can be predicted with 100% positive predictive power and a moderate (58.33%) negative predictive power when a combination of pretreatment tumor volume and changes in StO2 values pretreatment and immediately posttreatment was employed. Overall, our study indicates that US-PAI has the potential to provide label-free surrogate imaging biomarkers that can predict tumor growth rate in suboptimal therapy.
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The authors wish to make the following corrections to this paper [...].
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Developing a cancer theranostic nanoplatform with diagnosis and treatment capabilities to effectively treat tumors and reduce side effects is of great significance. Herein, we present a drug delivery strategy for photosensitizers based on a new liquid metal nanoplatform that leverages the tumor microenvironment to achieve photodynamic therapeutic effects in pancreatic cancer. Eutectic gallium indium (EGaIn) nanoparticles were successfully conjugated with a water-soluble cancer targeting ligand, hyaluronic acid, and a photosensitizer, benzoporphyrin derivative, creating EGaIn nanoparticles (EGaPs) via a simple green sonication method. The prepared sphere-shaped EGaPs, with a core-shell structure, presented high biocompatibility and stability. EGaPs had greater cellular uptake, manifested targeting competence, and generated significantly higher intracellular ROS. Further, near-infrared light activation of EGaPs demonstrated their potential to effectively eliminate cancer cells due to their single oxygen generation capability. Finally, from in vivo studies, EGaPs caused tumor regression and resulted in 2.3-fold higher necrosis than the control, therefore making a good vehicle for photodynamic therapy. The overall results highlight that EGaPs provide a new way to assemble liquid metal nanomaterials with different ligands for enhanced cancer therapy.
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BACKGROUND: Morbidity and mortality due to oral cancer in India are exacerbated by a lack of access to effective treatments amongst medically underserved populations. We developed a user-friendly low-cost, portable fibre-coupled LED system for photodynamic therapy (PDT) of early oral lesions, using a smartphone fluorescence imaging device for treatment guidance, and 3D printed fibreoptic attachments for ergonomic intraoral light delivery. METHODS: 30 patients with T1N0M0 buccal mucosal cancer were recruited from the JN Medical College clinics, Aligarh, and rural screening camps. Tumour limits were defined by external ultrasound (US), white light photos and increased tumour fluorescence after oral administration of the photosensitising agent ALA (60 mg/kg, divided doses), monitored by a smartphone fluorescence imaging device. 100 J/cm2 LED light (635 nm peak) was delivered followed by repeat fluorescence to assess photobleaching. US and biopsy were repeated after 7-17 days. This trial is registered with ClinicalTrials.gov, NCT03638622, and the study has been completed. FINDINGS: There were no significant complications or discomfort. No sedation was required. No residual disease was detected in 22 out of 30 patients who completed the study (26 of 34 lesions, 76% complete tumour response, 50 weeks median follow-up) with up to 7.2 mm depth of necrosis. Treatment failures were attributed to large tumour size and/or inadequate light delivery (documented by limited photobleaching). Moderately differentiated lesions were more responsive than well-differentiated cancers. INTERPRETATION: This simple and low-cost adaptation of fluorescenceguided PDT is effective for treatment of early-stage malignant oral lesions and may have implications in global health.
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Neoplasias de la Boca , Fotoquimioterapia , Ácido Aminolevulínico/uso terapéutico , Humanos , India , Neoplasias de la Boca/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
Photodynamic therapy (PDT) is a well-known cancer therapy that utilizes light to excite a photosensitizer and generate cytotoxic reactive oxygen species (ROS). The efficacy of PDT primarily depends on the photosensitizer and oxygen concentration in the tumor. Hypoxia in solid tumors promotes treatment resistance, resulting in poor PDT outcomes. Hence, there is a need to combat hypoxia while delivering sufficient photosensitizer to the tumor for ROS generation. Here we showcase our unique theranostic perfluorocarbon nanodroplets as a triple agent carrier for oxygen, photosensitizer, and indocyanine green that enables light triggered spatiotemporal delivery of oxygen to the tumors. We evaluated the characteristics of the nanodroplets and validated their ability to deliver oxygen via photoacoustic monitoring of blood oxygen saturation and subsequent PDT efficacy in a murine subcutaneous tumor model. The imaging results were validated with an oxygen sensing probe, which showed a 9.1 fold increase in oxygen content inside the tumor, following systemic administration of the nanodroplets. These results were also confirmed with immunofluorescence. In vivo studies showed that nanodroplets held higher rates of treatment efficacy than a clinically available benzoporphyrin derivative formulation. Histological analysis showed higher necrotic area within the tumor with perfluoropentane nanodroplets. Overall, the photoacoustic nanodroplets can significantly enhance image-guided PDT and has demonstrated substantial potential as a valid theranostic option for patient-specific photodynamic therapy-based treatments.