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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - NanoOxySens (Luminescent Polymer Nanoparticles with Aza-BODIPYs, Porphyrins and their Metal Complexes for FRET Mediated Ratiometric Oxygen Sensing)

Teaser

Summary

What is the problem/issue being addressed?
Detection of early stage of cancer and monitoring its response to a treatment is a major challenge in the research on diagnostics and therapy. One of the pathways which aid the early stage diagnosis of tumours is to detect and image the hypoxic regions in cells, which normally exist as oxygen gradients. The accurate detection and imaging of the molecular oxygen requires robust, safe and sensitive probes. Hence, the major challenge to be addressed here is to convert ultrabright dye-loaded polymeric nanoparticles into ratiometric sensors for the detection and imaging of hypoxic regions in cancer cells. Second, it is challenging to generate a stable oxygen gradient conditions for evaluation of the probes and modelling hypoxia conditions in tumours.

Why is it important for society?
The limitation and failure of early stage detection and diagnosis leads to increased risks of inefficient therapy of cancer patients, which constitute an important concern for the society. Hypoxia is a pathophysiological condition, commonly referred as a state of reduced oxygen tension, which is characteristic feature of tumours and other pathologies. Accurate detection and imaging of hypoxic regions can be a solution for the early stage diagnosis of various tumours. Here, we successfully demonstrated the FRET-based ultrabright polymeric nanoparticles for the ratiometric detection and imaging of oxygen gradients in cancer cells, which we believe can address the concern of early stage cancer diagnosis in the future and thus can improve the public health. The developed probes have a good potential for commercialization as high-tech products for biomedical applications.

What are the overall objectives?
The principal objective of the project was to develop an ultrabright polymeric nanoprobe for the FRET-mediated ratiometric detection of molecular oxygen. In particular, to build the nanoprobes we aimed to use (i) biocompatible polymer to address the issues of safety; (ii) high concentration with loaded fluorescent dyes with the bulky hydrophobic counterions to ensure efficient fluorescence and (iii) phosphorescent FRET acceptor to ensure ratiometric response to oxygen. The second objective is accurate detection of oxygen gradients in cancer cells. Specifically, we aimed to develop a simple and convenient microfluidic chamber with a stable oxygen gradient and compatible with fluorescence imaging of cancer cells and our polymeric nanoprobes.

Work performed

Our proposed objective was to design nanoscopic tools for detection of molecular oxygen and its gradients in cancer cells. As proposed, we developed a Forster Resonance Energy Transfer (FRET) based ratiometric oxygen sensor, using ultrabright polymeric nanoparticles based on a biocompatible polymer, a cyanine donor (BlueCy) dye with a fluorinated tetraphenyl borate counterion (TPB) and a porphyrin acceptor (PtOEP) as the oxygen sensitive moiety. The specific objectives were achieved through the following individual tasks of the work plan.
WP1: Development of fluorescent/phosphorescent polymeric nanoparticles with aza-BODIPY/porphyrin dyes
We started the project by selecting/synthesizing different acceptor (aza-BODIPY and porphyrins) and donor dyes (cyanines and tetrapheylethenes) for our FRET based nanoprobe and screened the best ones which meet the criteria related efficient FRET, good donor-acceptor spectral resolution and high sensitivity to molecular oxygen. The synthesized dyes were further loaded into polymeric nanoparticles (PMMA-MA NPs) through nanoprecipitation and characterized at the single particle level. Cyanine based donors (BlueCy-TPB) exhibited exceptional brightness of ca. 70 times higher compared to quantum dots (QD525).
As a continuation of the search for new donor dyes, we have also developed tetraphenyl-ethylene-based ionic aggregation-induced emission dyes (AIEgens) with bulky counterions. Encapsulation of these salts into PMMA-MA NPs revealed that bulky counterions ensure (i) formation of small (~50 nm) AIEgen-loaded polymeric NPs; (ii) good fluorescence quantum yield of encapsulated dyes (up to 30%); and (iii) NIR emission reaching 700 nm. Single-particle microscopy revealed that our 50-nm AIEgen-loaded PMMA-MA NPs were 6-fold brighter than the NIR emitting quantum dots (QD705). These NPs were found to be suitable for live cell imaging.
Among the two types of acceptors developed here we selected BlueCy-TPB as FRET donor for construction of the nanoprobes for oxygen sensing. As a phosphoresetn FRET acceptor, we first investigated aza-BODIPYs. However, their emission was found poorly sensitive to the molecular oxygen. Therefore, we focused on platinum complexes of porphyrins and found PtOEP and its long-wavelength analogue as promising candidates, showing strong dependence of its phosphorescence on oxygen concentrations.
WP2: Development of ratiometric dual color oxygen sensor
For our first probe, donor-acceptor NPs were prepared by encapsulating thousands of donors (BlueCy-TPB) per particle with small amount of acceptors (PtOEP) and observed efficient energy transfer from the donor to acceptor. We further found that the acceptor phosphorescence depended on oxygen in the solution, allowing its ratiometric detection at the single-particle level and in HeLa cells by optical microscopy. Based on a microfluidic chamber, we generated stable cellular oxygen gradients in cell culture, visualized by the nanoprobes. We also have successfully extended our design concept to (i) new near-infrared probe for molecular oxygen, which will be compatible with in vivo imaging, and (ii) ratiometric nanoprobe for the detection of endogenous nitric oxide (NO) in cells, which is another gas with important biological activity.
The results on AIEgen-based polymeric NPs were published (Nanoscale, 2019, 10.1039/c9nr04085d) and the oxygen sensor manuscript is ready for submission. We are also preparing two more manuscripts for publication based on this work. Apart from the manuscripts, the results of the Marie Curie project were disseminated as oral presentations at two international conferences (XVIIIth International Symposium on Luminescence Spectrometry, 2018 and European Materials Research Society, EMRS-2019) and as a poster at the local conference of the campus (JCI-2018).

Final results

The alarming statistics on the cancer and related diseases across the world, specifically in Europe is a matter of big concern. Detection of oxygen deficient regions in the tumor tissues are of primary importance for the early stage diagnosis of cancer diseases and designing the treatment strategy. In this respect, our research was focused on developing a sensing platform for the detection of molecular oxygen. So far, dye-loaded polymeric NPs have not been explored for designing nanoprobes for molecular oxygen. In particular, the light-harvesting strategy, where multiple energy donor dyes transfer energy to few oxygen-sensitive acceptors have not been realized. Here, based on this concept, we developed an ultrabright polymeric nanoprobes for the ratiometric detection and imaging of oxygen deficient areas in HeLa cells. Moreover, we proposed an original approach to generate an oxygen gradient in cell culture, which will help to validate new probes and to investigate the role of hypoxia in the development of diseases at the level of biological models.