Multimodal optical systems for clinical oncology

This thesis presents three multimodal optical (light-based) systems designed to improve the capabilities of existing optical modalities for cancer diagnostics and theranostics. Optical diagnostic and therapeutic modalities have seen tremendous success in improving the detection, monitoring, and treatment of cancer. For example, optical spectroscopies can accurately distinguish between healthy and diseased tissues, fluorescence imaging can light up tumours for surgical guidance, and laser systems can treat many epithelial cancers. However, despite these advances, prognoses for many cancers remain poor, positive margin rates following resection remain high, and visual inspection and palpation remain crucial for tumour detection. The synergistic combination of multiple optical modalities, as presented here, offers a promising solution. The first multimodal optical system (Chapter 3) combines Raman spectroscopic diagnostics with photodynamic therapy using a custom-built multimodal optical probe. Crucially, this system demonstrates the feasibility of nanoparticle-free theranostics, which could simplify the clinical translation of cancer theranostic systems without sacrificing diagnostic or therapeutic benefit. The second system (Chapter 4) applies computer vision to Raman spectroscopic diagnostics to achieve spatial spectroscopic diagnostics. It provides an augmented reality display of the surgical field-of-view, overlaying spatially co-registered spectroscopic diagnoses onto imaging data. This enables the translation of Raman spectroscopy from a 1D technique to a 2D diagnostic modality and overcomes the trade-off between diagnostic accuracy and field-of-view that has limited optical systems to date. The final system (Chapter 5) integrates fluorescence imaging and Raman spectroscopy for fluorescence-guided spatial spectroscopic diagnostics. This facilitates macroscopic tumour identification to guide accurate spectroscopic margin delineation, enabling the spectroscopic examination of suspicious lesions across large tissue areas. Together, these multimodal optical systems demonstrate that the integration of multiple optical modalities has potential to improve patient outcomes through enhanced tumour detection and precision-targeted therapies.Open Acces

The Development of Optical Nanosensor Technology for Single Cell Analysis

Advances in modern biosciences and optical biosensor technology have provided exciting new insights and capabilities. The integration of these fields has witnessed revolutionary advances, which include the development of optical nanosensors. Optical nanosensors are devices based on a direct spatial coupling between biologically active molecules and a signal transducer element interfaced to electronic equipment for signal amplification, acquisition and recording. Optical nanosensors consist of biorecognition molecules covalently immobilized onto the nanotips nanoscale optical fiber that serves as the transducing element. By combining the specificity of biorecognition molecules and the excellent sensitivity of laser-based optical detection, optical nanosensors are capable of detecting and differentiating biochemical constituents of complex systems enabling the provision of sensitive and specific identification of specific molecular events inside living cells. This work explores and focuses on the development and application of novel optical nanosensors for single living cell analysis. In this context, single cell analysis involves the application of optical nanosensor technology to observe and possibly map molecular events inside single living cells. Previous studies have focused on the bulk response of cells and this largely increases the probability of missing critical underlying mechanisms specific to the single cell. The ability to perform single cell analysis can dramatically improve our understanding of basic cellular processes e.g., signal transduction as well as improving our knowledge of the intracellular transport and the fate of therapeutic agents at the single cell level. This is important not only because of the capability to perform minimally invasive analysis, but also to overcome the problem of ensemble averaging. This capability to overcome ensemble averaging has the potential to yield new information that is not available from population averaged cellular measurements. This work involves the development and application of optical nanosensors for specific and sensitive chemical and protein analysis within single living cells. The ability of these sensors to successfully perform chemical and protein analysis at the single cell level, lay in their design specifications, size, specificity, sensitivity and eliminating interferences. With regard to their specifications, their size was in the nanometer regime, which is relative to the scale of a single mammalian cell (~ 10 µm) to allow non-invasive-to-minimally-invasive measurements in single living cells. In addition, they incorporated biological recognition molecules to achieve specificity and finally, near-field evanescent wave excitation and detection to achieve high sensitivity. High specificity and sensitivity allowed for precise and accurate identification of physicochemically detectable substances in complex matrices to eliminate any potential interference. The optical nanosensor intracellular measurement process is straightforward and begins with a sparsely distributed cell culture in a petri dish to allow viewing of single cells using an inverted fluorescence microscope. The optical nanosensor is secured onto the manipulating arms of the microscope and gently manipulated toward the single cell, interacting with the cell, penetrating but not disrupting cellular membranes. The optical nanosensor is briefly incubated in a single living cell and the laser is turned on and excitation light is launched into the optical nanosensor and propagated to the near field of the nanotip where the target analyte is excited by evanescent optical waves. The fluorescence signal generated when the target analyte is excited is collected by the optical set-up of the inverted fluorescence microscope, passes through spectral and spatial filters before detection with a sensitive photon counting photomultiplier tube (PMT). The PMT signal is amplified and recorded via a universal counter interfaced to a personal computer (PC). Data acquisition and recording are controlled using an integrated custom-written program, built on LabView platform. During in vitro and in vivo measurements, the optical nanosensor response is determined in terms of the sensitivity, specificity, linear dynamic range, response time, nanosensor stability, and reproducibility. In the course of experimental measurements, it was evident that optical nanosensors have characteristics including fast response times (msec range), sensitivity (pM range), selectivity, and excellent reproducibility. In addition to the above figures of merit, optical nanosensors demonstrated biocompatibility with no observed detrimental effects on the cell under investigation in control growth conditions. This demonstrated the utility of optical nanosensor technology for minimally invasive measurement of cellular reactions without altering or destroying the chemical make-up of the cell. This work also illustrates the potential of optical nanosensors in playing an important role in elucidating and enhancing our understanding of cell signaling and transduction pathways in real-time

Singlet Oxygen Dosimetry For Pleural Photodynamic Therapy

Photodynamic therapy (PDT) is a promising treatment modality that involves visible light and a photosensitizer to form reactive cytotoxic species, such as singlet oxygen in the case of type II PDT. Dosimetry of PDT has shown to be challenging due to the complex interactions between the key components of PDT: light, photosensitizer, and oxygen. Existing methods of quantifying dose involve monitoring one or two of these quantities. In conventional clinical settings, PDT is prescribed by the light fluence rate (mW/cm^2) and total light fluence (J/cm^2). However, many additional factors influence the effective ``dose\u27\u27 that is being delivered. Variations in photosensitizer uptake in tumors, tissue oxygenation, and light penetration in tissues of varying tissue optical properties affect the photodynamic efficiency. Using explicit dosimetry, reacted singlet oxygen is calculated based on the measured light fluence, photosensitizer concentration, and oxygen concentration. A macroscopic singlet oxygen model is used for explicit dosimetry, which involves various photochemical parameters. Relevant photochemical parameters for in vivo explicit dosimetry for a type II photosensitizer benzoporphyrin monoacid ring-A (BPD) were determined using a mouse model, and further validated using a study evaluating long term treatment outcome. Phantom studies were also performed to model the generation of singlet oxygen and compare it with direct measurements using singlet oxygen luminescence dosimetry (SOLD). Fluorescence spectroscopy methods were used to measure the drug concentration. Tissue optical properties were determined by measuring the light fluence and using the diffusion approximation for a point source at a fixed distance. Oxygenation was measured by using a phosphorescence-based probe to measure oxygen partial pressure. These in vivo and in-phantom models provide controlled environments where extensive explicit measurements can be performed to validate the model and recognize which aspects of explicit dosimetry are more critical to correctly correlate treatment outcome and the calculated dosimetric quantity. The light component of PDT dosimetry was investigated further in a clinical setting. Patients undergoing surgery for malignant pleural mesothelioma are treated with intraoperative PDT. The current treatment protocol for a clinical trial at the University of Pennsylvania involves monitoring light fluence at 8 discrete locations within the pleural cavity. Quantifying and planning treatment can be greatly improved by monitoring the light fluence throughout the entire treatment area in real-time. This work aims to provide details for singlet oxygen explicit dosimetry (SOED) to quantify the reacted singlet oxygen species during PDT in in vivo and in-phantom models. Furthermore, the light fluence modeling and calculation aspect of PDT dosimetry was developed and improved for an ongoing pleural PDT study at the University of Pennsylvania

Development of liposomal formulations for photodynamic therapy of cancer

Tese de mestrado integrado. Bioengenharia. Universidade do Porto. Faculdade de Engenharia. 201

Drug delivery in photodynamic therapy: From pharmaceutics to animal testing

S'ha estudiat el desenvolupament de fotosensibilitzadors i la seva formulació en teràpia fotodinàmica. S'han caracteritzat les propietats fotofísiques dels fotosensibilitzadors porficènics. S'han proposat diferents estratègies tals com la introducció de grups carboxilat en la perifèria o ions de metalls pesants en el nucli, per millorar el disseny de nous fotosensibilitzadors basats en el macrocicle porficènic. Entre ells, el temocè (m-THPPo), el porficè anàleg a la temoporfina, mostra excel•lents propietats fotofísiques, fotoestabilitat i alta eficàcia fotodinàmica. A causa de la seva alta hidrofobicitat, s'ha desenvolupat una formulació liposomal per a l'administració in vitro i in vivo del temocè. m-THPPo/DPPC/DMPG (1:67.5:7.5 relació molar) té alta eficiència d'encapsulació mantenint les seves propietats tant fotofísiques com a biològiques. El temocè liposomal va exhibir l'eficàcia fotodinàmica in vitro més alta per molècula internalitzada, sent un sistema d'administració de fàrmacs eficaç per a una estratègia in vivo dirigida a les cèl•lules tumorals. El temocè encapsulat en micel•les de Cremophor EL va mostrar una mínima internalització cel•lular. Consistentment, la formulació micel•lar va mostrar millor la resposta in vivo quan s'utilitza en un règim vascular. Amb la finalitat de minimitzar la internalització del fotosensibilitzador en les cèl•lules normals, es van proposar liposomes decorats amb lligands folat. Aquesta estratègia resulta en una internalització dues vegades major dels liposomes dirigits al receptor folat respecte a la corresponent formulació no específica. Finalment, han estat explorats nous models cel•lulars in vitro per a l'optimització dels processos amb oxigen singlet. Els cultius cel•lulars en 3D reprodueixen l'heterogeneïtat d'oxigen i fotosensibilitzador que està present en els teixits reals, proporcionant informació molt útil per interpretar i predir el resultat de la teràpia fotodinàmica. També s'ha demostrat la capacitat de desactivació de l'oxigen singlet d'antioxidants en un model ex vivo de pell porcina.Se ha estudiado el desarrollo de fotosensibilizadores y su formulación en terapia fotodinámica. Se han caracterizado las propiedades fotofísicas de los fotosensibilizadores porficénicos. Se han propuesto diferentes estrategias tales como la introducción de grupos carboxilato en la periferia o iones de metales pesados en el núcleo, para mejorar el diseño de nuevos fotosensibilizadores basados en el macrociclo porficénico. Entre ellos, el temoceno (m-THPPo), el porficeno análogo a la temoporfina, muestra excelentes propiedades fotofísicas, fotoestabilidad y alta eficacia fotodinámica. Debido a su alta hidrofobicidad, se ha desarrollado una formulación liposomal para la administración in vitro e in vivo del temoceno. m-THPPo/DPPC/DMPG (1:67.5:7.5 relación molar) tiene alta eficiencia de encapsulación manteniendo sus propiedades tanto fotofísicas como biológicas. El temoceno liposomal exhibió la eficacia fotodinámica in vitro más alta por molécula internalizada, siendo un sistema de administración de fármacos eficaz para una estrategia in vivo dirigida a las células tumorales. El temoceno encapsulado en micelas de Cremophor EL mostró una mínima internalización celular. Consistentemente, la formulación micelar mostró mejor la respuesta in vivo cuando se utiliza en un régimen vascular. Con el fin de minimizar la internalización del fotosensibilizador en las células normales, se propusieron liposomas decorados con ligandos folato. Esta estrategia resulta en una internalización dos veces mayor de los liposomas dirigidos al receptor folato respecto a la correspondiente formulación no específica. Por último, han sido explorados nuevos modelos celulares in vitro para la optimización de los procesos con oxígeno singlete. Los cultivos celulares en 3D reproducen la heterogeneidad de oxígeno y fotosensibilizador que está presente en los tejidos reales, proporcionando información muy útil para interpretar y predecir el resultado de la terapia fotodinámica. También se ha demostrado la capacidad de desactivación del oxígeno singlete de antioxidantes en un modelo ex vivo de piel porcina.The photosensitizer and formulation development in photodynamic therapy have been studied. They have been characterized the photophysical properties of new porphycene-based photosensitizers. Different strategies such as the introduction of carboxylate groups in the periphery or heavy metal ions in the core have been proposed for improving the design of novel photosensitizers based on the porphycene macrocycle. Among them, temocene (m-THPPo), the porphycene analogue to temoporfin, shows excellent photophysical properties, superior photostability and high photodynamic efficiency. Owing to its high hydrophobicity, a liposomal formulation has been developed for in vitro and in vivo administration of temocene. m-THPPo/DPPC/DMPG (1:67.5:7.5 molar ratio) yielded high encapsulation efficiency maintaining its photophysical and biological properties. Liposomal temocene exhibited the highest in vitro killing efficacy per uptaken molecule and they were an efficient drug delivery system for in vivo tumor cell targeting strategy. Temocene encapsulated in Cremophor EL micelles showed minimal cell internalization. Consistently, micellar formulation showed the best in vivo response when used in a vascular regime. In order to minimize the internalization of the photosensitizer in normal cells, liposomes decorated with folic acid ligands were proposed. This strategy leads to a 2-fold higher uptake of folate-targeted liposomes than the corresponding non-targeted formulation. Finally, new in vitro cellular models for a better optimization of singlet oxygen-involved processes were explored. 3D cellular cultures reproduced the oxygen and photosensitizer heterogeneity found in real tissues, providing useful information to interpret and predict the photodynamic therapy outcome. The singlet oxygen quenching ability of antioxidants in ex vivo porcine skin model has also been demonstrated

The Photosensitizer Temoporfin (mTHPC) – Chemical, Pre‐clinical and Clinical Developments in the Last Decade†‡

This review follows the research, development and clinical applications of the photosensitizer 5,10,15,20‐tetra(m‐hydroxyphenyl)chlorin (mTHPC, temoporfin) in photodynamic (cancer) therapy (PDT) and other medical applications. Temoporfin is the active substance in the medicinal product Foscan® authorized in the EU for the palliative treatment of head and neck cancer. Chemistry, biochemistry and pharmacology, as well as clinical and other applications of temoporfin are addressed, including the extensive work that has been done on formulation development including liposomal formulations. The literature has been covered from 2009 to early 2022, thereby connecting it to the previous extensive review on this photosensitizer published in this journal [Senge, M. O. and J. C. Brandt (2011) Photochem. Photobiol. 87, 1240–1296] which followed its way from initial development to approval and clinical application