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Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors. [ABSTRACT FROM AUTHOR]

Antimicrobial photodynamic therapy (aPDT) is an alternative to antibiotics that has potential for the treatment of chronic skin wounds, but requires improved, highly selective photosensitizer systems. Rhenium (Re)‐complex‐ and iridium (Ir)‐complex‐based phospholipid conjugates, as PDT‐functional building blocks for liposomes, are presented, and varying structural components and proportion of compounds are explored, including adjusting the cholesterol and polyethylene glycol (PEG)‐lipid contents, incorporating ethylenediaminetetraacetic acid (EDTA)‐lipid, and introducing the cationic lipid 1,2‐dioleoyl‐3‐trimethylammonium propane (DOTAP) to enhance their efficacy and selectivity in aPDT. Ir/Re‐liposomes have nanostructurally enhanced photoactivity compared to monomeric Ir/Re‐lipids. Ir‐liposomes exhibit stronger light absorption and higher emission generation (>threefold) than Re‐liposomes, resulting in superior efficacy against Staphylococcus aureus while maintaining better tolerability toward host cells. Formulations with higher cholesterol (40 mol%) and PEG‐lipid (5%) content demonstrate increased potency against S. aureus. The incorporation of EDTA‐lipid significantly enhances aPDT efficacy but also increases toxicity toward host cells. Incorporation of DOTAP alters the nanoparticles' surface charge, potentially improving their interaction with bacterial walls, but negatively impacts their stability, leading to aggregation of the nanoparticles. Ir‐HC demonstrates ideal characteristics (effectiveness, selectivity, and stability) for aPDT under the tested conditions, indicating the importance of the structural design of Re‐ and Ir‐complex liposomes for their selectivity in aPDT. [ABSTRACT FROM AUTHOR]

Photodynamic therapy is a treatment modality that can be used to treat various types of lesions. To produce cell death, reaching a certain threshold dose of reactive oxygen species (ROS) is required. The estimation of ROS production is of paramount importance to predict the depth of necrosis and to ensure that the volume to be treated receives doses higher than the threshold. In this study, we compared a theoretical model for PDT based on Monte Carlo simulations of light irradiance and rate equations with a rat liver model. At the end of the simulation, necrosis depths and volumes were estimated, as well as the photosensitizer (PS), oxygen, and ROS concentrations at each position of the treated area. From the in vivo study, we obtained the ROS concentration threshold of about 1 mM for Photogem in rat liver. This proposed method can be used for any PS or tissue, including tissues with multiple layers. The proposed method can be used to estimate parameters for any PS or tissue, including layered tissues, as long as their parameters are known. In addition, other protocols can be tested, or compared with the standard ones, providing the bases for analyzing a diverse range of photodynamic treatment scenarios. [ABSTRACT FROM AUTHOR]

Effective treatment of respiratory infections continues to be a major challenge. In high doses (≥160 ppm), inhaled Nitric Oxide (iNO) has been shown to act as a broad-spectrum antimicrobial agent, including its efficacy in vitro for coronavirus family. However, the safety of prolonged in vivo implementation of high-dose iNO therapy has not been studied. Herein we aim to explore the feasibility and safety of delivering continuous high-dose iNO over an extended period of time using an in vivo animal model. Yorkshire pigs were randomized to one of the following two groups: group 1, standard ventilation; and group 2, standard ventilation + continuous iNO 160 ppm + methylene blue (MB) as intravenous bolus, whenever required, to maintain metHb <6%. Both groups were ventilated continuously for 6 hours, then the animals were weaned from sedation, mechanical ventilation and followed for 3 days. During treatment, and on the third post-operative day, physiologic assessments were performed to monitor lung function and other significative markers were assessed for potential pulmonary or systemic injury. No significant change in lung function, or inflammatory markers were observed during the study period. Both gas exchange function, lung tissue cytokine analysis and histology were similar between treated and control animals. During treatment, levels of metHb were maintained <6% by administration of MB, and NO2 remained <5 ppm. Additionally, considering extrapulmonary effects, no significant changes were observed in biochemistry markers. Our findings showed that high-dose iNO delivered continuously over 6 hours with adjuvant MB is clinically feasible and safe. These findings support the development of investigations of continuous high-dose iNO treatment of respiratory tract infections, including SARS-CoV-2. [ABSTRACT FROM AUTHOR]

We present new approaches to treat both primary cutaneous and ocular melanoma, with potential to reduce metastatic spread In mouse models optical clearing combined with dual-photosensitizer PDT is used to treat primary cutaneous xenogafts. Two-photon PDT is used in ocular melanoma of extraocular (conjunctival) or intraocular (uveal) melanoma. Immune effects are investigated in immunocompetent mice using blood biomarkers and by tumor re-challenge. In vitro studies in pigmented and non-pigmented melanoma cells show melanin-mediated photosensitization as a novel mechanism of action of 2-γ PDT. Full-thickness primary tumor eradication has been achieved: ∼1 and 4 mm in immune deficient and competent mice, respectively. Marked reduction in metastatic tumor burden is observed in the latter. These studies reopen the possibility of PDT as an effective modality in melanoma. [ABSTRACT FROM AUTHOR]

Orthopedic surgery is frequently performed but currently lacks consensus and availability of ideal guidance methods, resulting in high variability of outcomes. Misdirected insertion of surgical instruments can lead to weak anchorage and unreliable fixation along with risk to critical structures including the spinal cord. Current methods for surgical guidance using conventional medical imaging are indirect and time-consuming with unclear advantages. The purpose of this study was to investigate the potential of intraoperative in situ near-infrared Raman spectroscopy (RS) combined with machine learning in guiding pedicular screw insertion in the spine. A portable system equipped with a hand-held RS probe was used to make fingerprint measurements on freshly excised porcine vertebrae, identifying six tissue types: bone, spinal cord, fat, cartilage, ligament, and muscle. Supervised machine learning techniques were used to train—and test on independent hold-out data subsets—a six-class model as well as two-class models engineered to distinguish bone from soft tissue. The two-class models were further tested using in vivo spectral fingerprint measurements made during intra-pedicular drilling in a porcine spine model. The five-class model achieved >96 % accuracy in distinguish all six tissue classes when applied onto a hold-out testing data subset. The binary classifier detecting bone versus soft tissue (all soft tissue or spinal cord only) yielded 100% accuracy. When applied onto in vivo measurements performed during interpedicular drilling, the soft tissue detection models correctly detected all spinal canal breaches. We provide a foundation for RS in the orthopedic surgical guidance field. It shows that RS combined with machine learning is a rapid and accurate modality capable of discriminating tissues that are typically encountered in orthopedic procedures, including pedicle screw placement. Future development of integrated RS probes and surgical instruments promises better guidance options for the orthopedic surgeon and better patient outcomes. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is a well-described technique that is primarily used for thin and superficial tumors. Despite the advantages of immune response upregulation, high complete response rates and excellent cosmetics and preservation of tissue structure, the limited light penetration into tissue complicates achieving full-thickness/large-volume treatment of pigmented, thicker, deep-seated or diffuse metastatic tumors1. Reported in vitro studies in melanoma and breast cancer cells show increased expression of major histocompatibility complex I after the combination of psoralen and X-radiation, which is representative of a potential immune response upregulation2. The aim here is to investigate the clinical photosensitizer Verteporfin (VP) as a radiosensitizer to achieve deep tissue penetration while retaining immune upregulation, and thereby enhance tumor-specific radiation responses in pancreatic cancer3 and melanoma4, which are aggressive tumors with high metastatic potential and low survival rates. In vitro studies were carried out with bioluminescent human pancreatic cancer (Panc-1-Luc-2) and murine melanotic melanoma (B16F10) cell lines. The cells were irradiated using 225 kVp (XRAD) or 6 MeV (Linac) X-ray beams, and the treatment response was measured by clonogenicity. In all cases, the presence of VP in the cells reduced survival compared with X-ray only controls: e.g. the group irradiated in the Linac, which includes a contribution from VP activation by X-ray induced Cherenkov light, reached up to 5 Logs of cell death after a single fraction of 10 Gy. In the ex-ovo model, duck eggs at 8 days post fertilization were engrafted with 15 µL (∼5 × 107) of Panc-1-Luc-2 or B16F10-Luc cells. Treatment was given 3 days later using topical VP administration with irradiation 1.5 h later. Combining a single fraction of 5 Gy with 5 µg/mL VP gave >2-fold reduction of the bioluminescence compared to <1 fold for radiation alone. After 3 daily fractions, the in ovo tumors treated with VP + radiation showed undetectable bioluminescence signal until sacrificed at 2 d after the last fraction. These studies are intended primarily to generate baseline response data for radiodynamic therapy with different nanoparticle formulations, including both lipid-porphyrin porphysomes and metal nanoclusters. ACKNOWLEDGEMENTS This work is supported by the Canadian Institutes of Health Research, The Terry Fox research institute and a Tri-Council NRFR grant. L. Pires is supported by the Banting Fellowship. [ABSTRACT FROM AUTHOR]

This study aimed to assess the photodynamic effect of Phonozen, a newly developed photosensitizer, on varied illumination wavelength of 405 nm and 664 nm in a mouse model with peritoneal carcinomatosis. Peritoneal carcinomatosis was induced by IP injection of 1 × 106 MKN45-luc cells to 9 week old female athymic nude mice. Ten days after induction, PDT was performed on 60 mice injecting Phonozen 3 hours before PDT. Twenty mice for each group underwent intra-peritoneal catheterization and underwent (A)no irradiation, irradiation with (B) 405nm, and (C) 664 nm for 1hour, delivering 50J per mouse. Fifteen of each group were observed for 20 weeks for survival analysis and five of each group were sacrificed 2 days after for quantitative assessment of necrosis status. Both irradiation approach with 405 nm and 664 nm significantly prolonged the survival of PC mice over control group. No survival difference were observed between two irradiated group. Semiquantitative assessment showed significantly higher proportion of responder following irradiation of 405 nm than 664 nm. Phonozen shows effective photodynamic tumoricidal effect. Activated by same power, 405 nm is more effective than 664nm for treatment of PC mice model [ABSTRACT FROM AUTHOR]

Melanoma is the most aggressive type of skin cancer, with significant risk of fatality. Due to its pigmentation, light-based imaging and treatment techniques are limited to near the tumor surface, which is inadequate, for example, to evaluate the microvascular density that is associated with prognosis. White-light diffuse reflectance spectroscopy (DRS) and near-infrared optical coherence tomography (OCT) were used to evaluate the effect of a topically applied optical clearing agent (OCA) in melanoma in vivo and to image the microvascular network. DRS was performed using a contact fiber optic probe in the range from 450 to 650 nm. OCT imaging was performed using a swept-source system at 1310 nm. The OCT image data were processed using speckle variance and depth-encoded algorithms. Diffuse reflectance signals decreased with clearing, dropping by ∼90% after 45 min. OCT was able to image the microvasculature in the pigmented melanoma tissue with good spatial resolution up to a depth of ∼300 μm without the use of OCA; improved contrast resolution was achieved with optical clearing to a depth of ∼750 μm in tumor. These findings are relevant to potential clinical applications in melanoma, such as assessing prognosis and treatment responses. Optical clearing may also facilitate the use of light-based treatments such as photodynamic therapy. [ABSTRACT FROM AUTHOR]

Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogen's cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment. [ABSTRACT FROM AUTHOR]

Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill. [ABSTRACT FROM AUTHOR]

Treatment using light-activated photosensitizers (photodynamic therapy, PDT) has shown limited efficacy in pigmented melanoma, mainly due to the poor penetration of light in this tissue. Here, an optical clearing agent (OCA) was applied topically to a cutaneous melanoma model in mice shortly before PDT to increase the effective treatment depth by reducing the light scattering. This was used together with cellular and vascular-PDT, or a combination of both. The effect on tumor growth was measured by longitudinal ultrasound/photoacoustic imaging in vivo and by immunohistology after sacrifice. In a separate dorsal window chamber tumor model, angiographic optical coherence tomography (OCT) generated 3D tissue microvascular images, enabling direct in vivo assessment of treatment response. The optical clearing had minimal therapeutic effect on the in control, non-pigmented cutaneous melanomas but a statistically significant effect (p < 0.05) in pigmented lesions for both single- and dual-photosensitizer treatment regimes. The latter enabled full-depth eradication of tumor tissue, demonstrated by the absence of S100 and Ki67 immunostaining. These studies are the first to demonstrate complete melanoma response to PDT in an immunocompromised model in vivo, with quantitative assessment of tumor volume and thickness, confirmed by (immuno) histological analyses, and with non-pigmented melanomas used as controls to clarify the critical role of melanin in the PDT response. The results indicate the potential of OCA-enhanced PDT for the treatment of pigmented lesions, including melanoma. [ABSTRACT FROM AUTHOR]

Availability of organs is a limiting factor for lung transplantation, leading to substantial mortality rates on the wait list. Use of organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), would increase organ donation, but these organs are generally not offered for transplantation due to a high risk of transmission. Here, we develop a method for treatment of HCV-infected human donor lungs that prevents HCV transmission. Physical viral clearance in combination with germicidal light-based therapies during normothermic ex-vivo Lung Perfusion (EVLP), a method for assessment and treatment of injured donor lungs, inactivates HCV virus in a short period of time. Such treatment is shown to be safe using a large animal EVLP-to-lung transplantation model. This strategy of treating viral infection in a donor organ during preservation could significantly increase the availability of organs for transplantation and encourages further clinical development. Organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), are not offered for transplantation due to a high risk of transmission. Here, Galasso et al. develop a method for treatment of HCV-infected human donor lungs that is safe and prevents HCV transmission in the pig model. [ABSTRACT FROM AUTHOR]

Transmission of latent human cytomegalovirus (HCMV) via organ transplantation with post-transplant viral reactivation is extremely prevalent and results in substantial adverse impact on outcomes. Therapies targeting the latent reservoir within the allograft to mitigate viral transmission would represent a major advance. Here, we delivered an immunotoxin (F49A-FTP) that targets and kills latent HCMV aiming at reducing the HCMV reservoir from donor lungs using ex-vivo lung perfusion (EVLP). HCMV seropositive human lungs were placed on EVLP alone or EVLP + 1mg/L of F49A-FTP for 6 hours (n = 6 , each). CD14+ monocytes isolated from biopsies pre and post EVLP underwent HCMV reactivation assay designed to evaluate viral reactivation capacity. Off-target effects of F49A-FTP were studied evaluating cell death markers of CD34+ and CD14+ cells using flow cytometry. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. We demonstrate that lungs treated ex-vivo with F49A-FTP had a significant reduction in HCMV reactivation compared to controls, suggesting successful targeting of latent virus (76% median reduction in F49A-FTP vs 15% increase in controls, p = 0.0087). Furthermore, there was comparable cell death rates of the targeted cells between both groups, suggesting no off-target effects. Ex-vivo lung function was stable over 6 hours and no differences in key inflammatory cytokines were observed demonstrating safety of this novel treatment. Ex-vivo F49A-FTP treatment of human lungs targets and kills latent HCMV, markedly attenuating HCMV reactivation. This approach demonstrates the first experiments targeting latent HCMV in a donor organ with promising results towards clinical translation. [ABSTRACT FROM AUTHOR]

Abstract This study describes non-invasive photoacoustic imaging to detect and monitor the growth of conjunctival melanomas in vivo. Conjunctival melanomas were induced by injection of melanotic B16F10 cells into the subconjunctival space in syngeneic albino C57BL/6 mice. Non-invasive in vivo photoacoustic tomography was performed before, and after tumor induction up to 2 weeks. Spectral unmixing was performed to determine the location and to assess the distribution of melanin. The melanin photoacoustic signal intensity was quantified from the tumor-bearing and control eyes at all timepoints. For postmortem validation, total tumor and melanotic tumor volumes were measured using H&E stained tumor sections and were compared to in vivo photoacoustic imaging measurements. Photoacoustic imaging non-invasively detected eyes bearing conjunctival tumors of varying sizes. The melanin signal was detected as early as immediately following injection of melanotic tumor cells. Changes in tumor size over time were assessed with changes in the volume and intensity of the melanin signal. Four growing tumors and one regressing tumor were observed. Three tumors without significant change in signal intensity over time were observed, showing variable growth. Photoacoustic melanin signal on the last day of in vivo imaging correlated with postmortem total tumor volume (R2 = 0.81) and melanotic tumor volume (R2 = 0.80). The results of our study show that actively growing conjunctival melanomas can be quantified in a non-invasive manner using in vivo photoacoustic tomography. The photoacoustic melanin signal intensity correlated with total and melanotic tumor volume. This novel in vivo imaging platform may help to assess new treatment modalities to manage ocular tumors. Highlights • Non-invasive in vivo photoacoustic imaging was used to detect and monitor conjunctival melanomas. • Melanin content in tumors was quantified using photoacoustic tomography. • Melanin signal intensity correlated with histomorphometry. • In vivo quantification of melanin signal over time showed various patterns of tumors growth. [ABSTRACT FROM AUTHOR]

Texture analyses of optical coherence tomography (OCT) images have shown initial promise for differentiation of normal and tumor tissues. This work develops a fully automatic volumetric tumor delineation technique employing quantitative OCT image speckle analysis based on Gamma distribution fits. We test its performance in-vivo using immunodeficient mice with dorsal skin window chambers and subcutaneously grown tumor models. Tumor boundaries detection is confirmed using epi-fluorescence microscopy, combined photoacoustic-ultrasound imaging, and histology. Pilot animal study of tumor response to radiotherapy demonstrates high accuracy, objective nature, novelty of the proposed method in the volumetric separation of tumor and normal tissues, and the sensitivity of the fitting parameters to radiation-induced tissue changes. Overall, the developed methodology enables hitherto impossible longitudinal studies for detecting subtle tissue alterations stemming from therapeutic insult., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Melanoma is the most aggressive type of skin cancer and a relevant health problem due to its poor treatment response with high morbidity and mortality rates. This study, aimed to investigate the tissue changes of an improved photodynamic therapy (PDT) response when combined with optical clearing agent (OCA) in the treatment of cutaneous melanoma in mice. Photodithazine (PDZ) was administered intraperitoneally and a solution of OCA was topically applied before PDT irradiation. Due to a resultant refractive index matching, OCA-treated tumors are more optically homogenous, improving the PDT response. Raman analysis revealed, when combined with OCA, the PDT response was more homogenous down to 725 µm-depth in thickness., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Melanoma is the most aggressive skin cancer type. It is characterized by pigmented lesions with high tissue invasion and metastatic potential. The early detection of melanoma is extremely important to improve patient prognosis and survival rate, since it can progress to the deadly metastatic stage. Presently, the melanoma diagnosis is based on the clinical analysis of the macroscopic lesion characteristics such as shape, color, borders following the ABCD rules. The aim of this study is to evaluate the time-resolved fluorescence lifetime of NADH and FAD molecules to detect cutaneous melanoma in an experimental in vivo model. Forty-two lesions were analyzed and the data was classified using linear discriminant analysis, a sensitivity of 99.4%, specificity of 97.4% and accuracy of 98.4% were achieved. These results show the potential of this fluorescence spectroscopy for melanoma detection.

Pythiosis is an infectious disease caused by Pythium insidiosum, a fungus-like organism. Due to the lack of ergosterol on its cell membrane, antibiotic therapy is ineffective. The conventional treatment is surgery, but lesion recurrence is frequent, requiring several resections or limb amputation. Photodynamic therapy uses photo-activation of drugs and has the potential to be an attractive alternative option. The in vitro PDT response on the growing of Pythium insidiosum culture was investigated using three distinct photosensitizers: methylene blue, Photogem, and Photodithazine. The photosensitizer distribution in cell structures and the PDT response for incubation times of 30, 60, and 120 minutes were evaluated. Methylene blue did not penetrate in the pathogen's cell and consequently there was no PDT inactivation. Photogem showed heterogenous distribution in the hyphal structure with small concentration inside the cells. Porphyrin-PDT response was heterogenous, death and live cells were observed in the treated culture. After 48 hours, hyphae regrowth was observed. Photodithazine showed more homogenous distribution inside the cell and with the specific intracellular localization dependent on incubation time. Photodithazine first accumulates in intracellular vacuoles, and at incubation times of one hour, it is located at all cell membranes. Higher inhibition of the growing rates was achieved with Photodithazine -PDT, over 98%. Our results showed that the photosensitizers that cross more efficiently the Pythium insidiosum membranes are able to cause extensive damage to the organism under illumination and therefore, are the best options for clinical treatment.