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).
