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Controlled Singlet Oxygen Release Photosensitizers in Photodynamic Therapy

Periodic Reporting for period 1 - CONSORT (Controlled Singlet Oxygen Release Photosensitizers in Photodynamic Therapy)

Reporting period: 2015-09-15 to 2017-09-14

The primary goal of CONSORT was to develop an innovative approach towards generation of singlet oxygen for photomedical therapies, such as photodynamic therapy (PDT), through introduction of a new type of photosensitizers. These molecular systems will be capable to generate singlet oxygen upon irradiation, store it by means of binding to special molecular subunits and slowly release singlet oxygen in biological media on a timescale from several hours to several days after light irradiation treatment. Delayed singlet oxygen formation will be demonstrated in cancer cells after the light treatment using in vitro models. The secondary objective of this project is to optimize the efficiency of novel photosensitizers to allow for increased therapeutic effect and reduced side-effects with respect to conventional PDT. Overall, this project will deliver a new means to treat cancer and other malignancies.

To test the hypothesis of this project, a series of research objectives were formulated and reached:
(i) Synthetic methods to access porphyrins with chemically bound oxygen trapping subunits will be developed;
(ii) Proof-of-concept studies will be performed to validate the use of thermal singlet oxygen release and optimize the efficiency of corresponding molecular systems;
(iii) Biopolymers bearing chemically modified with oxygen trapping subunits in the polymer chain and decorated with conventional PDT sensitizer will be developed;
(iv) In vitro screening protocols for PDT in living cells using the concept of delayed singlet oxygen release will be performed.
The activities of the project were organized in four work packages:

WP1: Chemical synthesis of porphyrin-photosensitizers containing oxygen trapping subunits
Optimization of the synthetic protocol and scale-up of the synthesis, investigation of the optical properties and structural data for target compounds has been achieved.
The fellow has developed a facile and reliable synthetic approach towards porphyrins, which bear 4-8 oxygen trapping subunits. A library was prepared for in vitro cell tests. Additionally, a new type of singlet oxygen sensitizers based on heavy-atom free BODIPY-anthracene dyads was discovered. Optical properties of the materials were studied in collaboration with Prof. Laquai (KAUST) to reveal photo-excited states dynamics, responsible for the generation of singlet oxygen. Structural data have been obtained unravelling the molecular structures for 16 newly prepared photosensitizers. This data has been analysed in conjunction with optical properties to establish structure-properties relationships for optimizing photosensitization efficiencies. WP2: WP2 aims at the evaluation of the most efficient photosensitizers in terms of singlet oxygen formation yield
The fellow has performed research on singlet oxygen sensing using optical spectroscopy to elucidate the efficiencies of the photosensitized and thermal singlet oxygen generation by the developed new sensitizer systems.

The fellow has optimized singlet oxygen sensing procedures, applying chemical acceptor molecules able to react with singlet oxygen and give optical response which can be monitored by UV-Vis and fluorescence spectroscopy in order to determine the timescale and efficiency of the process. The effects of environmental polarity and temperature on were studied using this technique and showed that porphyrins with singlet oxygen traps are capable to bind singlet oxygen reversibly and, that the release can be controlled by temperature. A formation of 2-pyridone endoperoxides, stable at room temperature, which selectively decompose at moderate heating (40 °C) on a timescale of several hours has been found. Selective generation of singlet oxygen in either non-polar or polar media has been discovered for BODIPY-anthracene dyads. Additionally, a fluorescent response of toward self-sensitized singlet oxygen, allowing for visualization of the photosensitization process, was discovered.

WP3: Polymeric material modified with oxygen trapping subunits and conventional PDT sensitizer
The fellow has explored chemical modification of chitosan biopolymer with naphthalene and 2-pyridone derivatives capable of reversible singlet oxygen binding. These materials have been characterized and their singlet oxygen generation ability has been studied. Thus obtained polymers were further modified with porphyrin photosensitizer to obtain cross-linked hydrogel.

WP4: In vitro studies
The newly prepared materials were studied using in vitro cell models to evaluated cytotoxicity and demonstrate the effect of delayed release of singlet oxygen. This has been coordinated with the company Biolitec Pharma and collaborating with the group of Prof. Boyle at the University of Hull, UK. I These derivatives were studied with human cancer cells. A proof-of-principle for delayed singlet oxygen release from the endoperoxide moieties has been demonstrated. Additionally, novel singlet oxygen-responsive photosensitizers have been applied to study the effects of oxidative stress on the cells.

Deliverables and milestones achieved:
WP1: A general method of synthesis of porphyrin photosensitizers bearing oxygen trapping subunits has been developed and target compounds for in vitro study have been prepared on large scale. Synthetic protocols for further publication and potential commercialization have been documented. Novel photosensitizers based on heavy atom-free donor-acceptor dyads have been discovered. Structural data, including several X-ray structures has been obtained. Data on o
The developed molecular systems and materials are capable of the controlled reversible binding of singlet oxygen. This allows for the storage of oxygen and its transport to a target site, e.g. localization in living cells. Oxygen can then be released under a stimuli which raises the local temperature within the shuttle. Chemical tuning of the system subunits allows to adjust the temperature range of the release process, while the activation stimulus provides spatiotemporal control over the process. This allows for precisely controlled generation of reactive oxygen species in target cells. Such systems are highly important from a fundamental point of view as it provide a new paradigm for the design of artificial systems mimicking the process of oxygen transport and activation by blood cells. Alternatively, the development of new photomedical treatments causing less side effects becomes feasible.
Reactive oxygen species can be used to combat microorganism and malignant tissue