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BrightSens Report Summary

Project ID: 648528
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - BrightSens (Ultrabright Turn-on Fluorescent Organic Nanoparticles for Amplified Molecular Sensing in Living Cells)

Reporting period: 2015-06-01 to 2016-11-30

Summary of the context and overall objectives of the project

The field of fluorescent nanoparticles (NPs) for bioimaging is growing exponentially. However, the research has been focused on inorganic nanoparticles, mainly quantum dots, which are not biodegradable and composed of toxic elements. Therefore, synthesis of both ultrabright and biodegradable nanoparticles constitutes the first challenge of the project. Moreover, not much is known on how to control fluorescence of nanoparticles by external stimuli, so that their application for detection of biomolecules is still in its infancy. Thus, the second challenge is to establish mechanisms of signal amplification, where a single external molecular stimulus is converted into a strong fluorescence response of a nanoparticle. Finally, detecting and imaging individual biomolecules at work directly in living cells is still far from realization. The third challenge is to develop nanoparticle probes with amplified turn-on response to molecular targets at the surface and in cytosol of living cells, with particular focus on cancer markers.

Three objectives of BrightSens are to address the three challenges described above: (1) To obtain fluorescent organic nanoparticles with high brightness and collective FRET to a single acceptor by resolving fundamental problems of dye self-quenching and energy transfer on the nanoscale. (2) To develop nanoparticle probes that turn-on >100 fluorescent dyes in response to single molecular targets (membrane receptors and mRNA). (3) To validate the nanoprobes in 2D and 3D cell cultures for ultrasensitive detection of cancer markers at the cell surface (integrin, EGFR and folate receptors) and in the cytosol (mRNA of survivin and Bcl-2).

As an impact of BrightSens project on society, several points should be mentioned. First, we strive to develop biodegradable/biocompatible nanoparticles, which are expected to have minimal danger for human health and ecology. Therefore, we aim to move towards safer nanotechnology. Second, our new probes for detection of cancer markers are expected to become tools for cancer diagnostics and personalised medicine. These probes are expected to simplify and decrease costs of protocols for detection of these markers. Thus, ultimately, BrightSens will have an important impact on human health.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Within Work package 1 (WP1, Fluorescent Organic Nanoparticles), we developed new fluorescent nanoparticles (NPs). (1) We obtained very small and ultrabright nanoparticles based on ion association of rhodamine dyes with bulky hydrophobic counterions. These particles are much brighter than quantum dots having small size of 10-20 nm (Shulov et al, Nanoscale, 2015). Their stability in cellular environment depends of the fluorination level of the counterion. (2) In the second approach, small size of NPs was achieved through assembly of amphiphilic cyanine dyes into micelles. However, the capacity of the obtained cyanine amphiphiles to form micelles was weak (high critical micellar concentration) and the assembly resulted in strong self-quenching of dyes. Remarkably, bulky hydrophobic counterions (of tetraphenylborate family) induced strong assembly of cyanine amphiphiles into ultrasmall micellar NPs (10 nm) exhibiting efficient fluorescence (Shulov et al, Chem. Commum, 2016). (3) However, in both previous examples stability in biological media was limited, therefore, we aimed to obtain polymerized fluorescent micelles. To this end we followed an original design, where PEGylated cyanine bis-azides formed a covalently attached “corona” on micelles of amphiphilic calixarene bearing alkyne groups. The obtained protein-sized (7 nm) fluorescent NPs were ~2-fold brighter than quantum dots-585. FRET studies suggested their high stability in aqueous and organic media as well as in living cells, where they showed excellent imaging contrast (Shulov et al, Angew. Chem. Int. Ed., 2016). (4) We worked on improving the surface properties of our previously developed dye-loaded polymer NPs. Using Pluronic block copolymer surfactant, we were able strongly decrease non-specific interactions of NPs with cells. We showed that this surfactant is able to bind strongly to particle surface, which is an interesting alternative to covalent modification of NPs with PEG groups (Heimburger et al, in preparation). (5) Our counterion approach for encapsulation of rhodamine dyes was extended to cyanines, which allowed varying the colour of NPs from green till near infrared. Hydrophobic counterion was crucial to prevent cyanine self-quenching of cyanines inside polymer matrix and ensure formation of small particles. These multicolour NPs was applied for colour coding of living cells in vitro and in vivo (Andreiuk et al, submitted). (6) We discovered some NPs that undergo highly efficient FRET (Trofymchuk et al, submitted), which is important for development of FRET-based nanoprobes in WP2.

Within WP2 on Nanoprobes (which started recently), we worked on development of model FRET based nanoparticle probes as well as on molecular probes that can be further grafted to NPs. (1) FRET was first investigated in dye-doped silica NPs (in collaboration with Prof. Luca Prodi, University of Bologna). Using specially designed BHQ-based quenchers, we obtained NPs that change their emission lifetime in response to the reductive environment inside the cells (Petrizza et al, RSC Adv, 2016). (2) In collaboration with Dr. J. Goetz and N. Anton (University of Strasbourg, France), we showed that FRET within near-infrared dyes inside NPs can be used for quantitative imaging of particle integrity. We found that despite their liquid nature, lipid NPs preserve their integrity in blood and can enter tumours in nearly intact form (Bouchaala, et al, J. Control. Release, 2016). (3) To target membrane receptors, we started with a carbetocin, a ligand for G-protein coupled receptor, previously studied by us earlier. This ligand has been coupled to a molecular rotor, in collaboration with Dr. Y. Kovtun (Institute of Organic Chemistry, Ukraine) and Dr. D. Bonnet (University of Strasbourg, France). The obtained conjugate showed turn-on response in the presence of target oxytocin receptor, which appears as molecular prototype of future nanoprobes (Karpenko et al, J. Mater. Chem. C, 2016). (4) In order to obtain a powerful FRET acceptor for NPs, we synthesized octamer of squaraine dye, which showed turn-on response to environment polarity (Ashokkumar et al, in preparation).

An important review on dye-loaded polymer nanoparticles was prepared and published (Reisch and Klymchenko, Small, 2016), which is classified as “highly cited paper” by ISI Web of Science.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

I. Progress beyond the state of the art in the field of fluorescent nanoparticles.
1) The role of counterion fluorination in preventing dye self-quenching was described for the first time (Shulov et al, Nanoscale, 2015).
2) Hydrophobic counterions were proposed for the first time as agents for self-assembly of fluorescent amphiphiles into ultra-small NPs (Shulov et al, Chem. Commum, 2016).
3) An original concept of nanoparticle design was proposed, where micellar nanoparticles are cross-linked by cyanine corona. These are unique nanoparticles, presenting small size, fluorogenic behaviour and high brightness in the ON-state (Shulov et al, Angew. Chem. Int. Ed., 2016).
4) Stability of Pluronic adsorption on polymer NPs surface was addressed (Heimburger et al, in preparation).
5) For the first time, fluorinated counterions were proposed for encapsulation of cyanines inside polymer NPs. Moreover, we proposed an original approach for colour coding of cells and their further tracking in vitro and in vivo (Andreiuk et al, submitted).
6) Unprecedented FRET efficiency in nanoparticles was discovered (Trofymchuk et al, submitted).

All these developments will have direct impact on the next steps of the project, notably for preparation of nanoparticle probes. Moreover, these findings propose new routes to ultrabright fluorescent NPs and stimulate the research on fluorescent organic NPs. Due to all-organic nature of our NPs, we propose eco-friendly nanoparticles, which will constitute the step towards safer nanotechnology with corresponding societal implications.

II. Progress beyond the state of the art in the field of fluorescent probes.
1) Fluorescent turn-on nanoparticle probes for lifetime imaging of reductive environment in living cells were developed (Petrizza et al, RSC Adv, 2016).
2) For the first time, quantitative analysis of integrity of lipid NPs in blood circulation and tumours was estimated using ratiometric near-infrared FRET imaging (Bouchaala, et al, J. Control. Release, 2016).
3) Molecular rotor dyes were applied for the first time to design turn-on probes for G-protein coupled receptors (Karpenko et al, J. Mater. Chem. C, 2016).
4) Original dendrimeric dye presenting turn-on response to environment polarity was developed (Ashokkumar et al, in preparation).

The obtained results provide important clues to development of nanoparticle-based probes for receptors and nucleic acids within BrightSens. The socio-economic impact is expected at the later steps as soon as we succeed in the development of these probes, because they can become important tools for ultrasensitive detection of cancer markers.

Related information

Record Number: 198656 / Last updated on: 2017-05-23
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