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Bio-conjugatable and Bio-responsive Near Infrared Fluorochromes

Periodic Reporting for period 1 - NIR-BIO-FLU (Bio-conjugatable and Bio-responsive Near Infrared Fluorochromes)

Reporting period: 2016-03-01 to 2018-02-28

The development of novel imaging agents to assist in the detection of several kinds of cancer diseases is one actual challenge of the scientific community due cancer remains as the major public health issue in Europe because of its high prevalence, morbidity, and mortality. In this aspect, near-infrared (NIR) fluorescence imaging has been clinically used in cancer targeting and imaging due to deep photon penetration in tissue (10-15 nm), minimizes photo-damage to biological samples and produces low background auto-fluorescence from biomolecules present in living systems. , It supposes a huge advance in clinical NIR-fluorescence imaging could be exploited to assist surgeons to distinguish between malignant and normal tissues in the challenging environment of an operating theatre. For example, over 50% of all cancerous conditions are first treated with surgery with the only guide available to the surgeon being their experience. It is currently not possible to differentiate between non-diseased lymph nodes from those that contain metastatic cancer lesions. If improvements to surgical outcomes utilizing the advantages of NIR-fluorescence could be achieved the improvement of societal health in Europe would be far-reaching.
Boron-chelated aza-dipyrromethenes (NIR-AZA) is an easily synthesizable novel NIR-fluorophore class with suitable properties, as strong absorptions and emissions within the NIR spectral, highly photostability and good quantum yields, to be employed in biological imaging (Figure 1). Consider the above mentioned, the objectives of this project involve:

1. Synthesis and characterization of new bio-responsive and bionconjugable NIR-AZA

2. Desing and synthesis of NIR-AZA to assist in the lymphnode detection.

3. Develop new cell membrane responsive NIR-Fluorescent probe

4. Delopvement of new strategies for the synthesis of NIR-AZA
During the course of this project, we carried out the synthesis and characterization of new bio-responsive and bioconjugable fluorophores based on aza-dipyrromethene BF2 complex (Figure 2). The rational design of this fluorophores allowed the control of fluorescence emission in function of pH of the media. These new fluorophores showed a pKa value centred at 5.5 making suitable for specific recognition at endogenous event. As we expected, at extracellular pH (pH = 7.4) the probe presented an ionized phenolate form, causing the fluorescence quenching. However, upon protonation to the neutral phenol species as established, providing the NIR emission. In addition, the new bio-responsive NIR-fluorophore were successfully attached covalently to RGD peptide-based targeting integrins, which over-expression has been found in various types of colon tumours, resulting in a potential tool for real-imaging fluorescence in colon cancer (Figure 3).

At the same time that we carried out the main objective of the project, we developed a new water-soluble NIR-AZA fluorophore with excellent photophysical characteristics including substituent determined emission maxima between 675 and 800 nm, high quantum yields and exceptional photostability (Figure 4). These properties have allowed it to be directly translatable from in vitro live cell imaging to in vivo imaging including the potential for clinical applications in fluorescence-guided surgery. The specifical design of this water-soluble NIR-AZA fluorophore with inherent absorption and emission max wavelengths in the 695-730 nm range can be utilised for real-time cell membrane imaging with the emission signal being turned on due to cell membrane interaction and when formulated using a clinically used insipient can give rise to micellar confined J aggregates with emission wavelength maxima beyond 800 nm which be used for in vivo small animal imaging and lymph node mapping using clinical imaging instrumentation.

To expand our knowledge in the uses f NIR-fluorophores for real-imaging fluorescence, we developed novel dimethyl-amino substituted NIR-AZA (Figure 5) with photophysical properties in the spectral region required for
small animal research and clinical use(> 780 nm). Preliminary assessments with commonly used research and clinical instrumentation clearly illustrate its suitability for use with these imaging devices. Positive fluorescent tattooing and lymphatic imaging results in ex vivo human colonic tissue specimens show, for the first time, the clinical potential of the NIR-AZA fluorophore class. This proof-of-concept our study illustrates how dimethylamino substitution of NIR-AZA fluorophores can provide the spectral wavelengths from which new medical and surgical imaging procedures may emerge.

Finally, we developed a new adaptable and versatile approach for the synthesis of new water-soluble 1,9-alkyl NIR-AZA with low lipophilicity, suitable for being covalently attached to different biomolecules, making them an innovative tool for real-time imaging (Figure 6). Considering this aspect, the development of new strategies to obtain new NIR-fluorophores, which allow us to control and/or modify their behaviour, is one of the current challenges.The advances in this field would mean an improvement in the tools for detection and treatment of different types of cancer.These exceptional outcomes will be communicated in high-impact peer reviewed journals such as Nature Commun., Angew. Chemie Int. Ed., J. Am. Chem. Soc, or Chem. Eur. Jo, where the O'Shea group regularly publish their achievements. In accordance with RCSI policy on Open Acess, they will be deposit in the repository. Dr Durán will attend conferences within specific area, such as Photo IUPAC celebrated on July of 2018 (July).
The present project was perfectly integrated within the priorities of the Horizon 2020, whose main objectives are focused on improving knowledge of the causes and underlying mechanisms in diseases as well as perfect the ability to monitor the state healthy, prevent, detect and treat diseases by optimizing their diagnosis and prognosis. So, considering this point of view, the results obtained during the development of this project, these will contribute to the improvement of the detection and treatment of colon cancer, reducing its impact on the health of the population.
Figure 4. Cell-membrine responsive NIR-Aza
Figure 3. Bionconjugable NIR-Aza
Figure 6. Water soluble 1,9-dialkyl NIR-Aza
Figure 1. NIR-Aza fluorophore
Figure 5. BF2-azadipyrromethene NIR-emissive fluorophores with research and clinical potential
Figure 2. Design of bioconjugabe and bioresponsive NIR-Azas