Skip to main content

Ultrabright Turn-on Fluorescent Organic Nanoparticles for Amplified Molecular Sensing in Living Cells

Periodic Reporting for period 3 - BrightSens (Ultrabright Turn-on Fluorescent Organic Nanoparticles for Amplified Molecular Sensing in Living Cells)

Reporting period: 2018-06-01 to 2019-11-30

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.
Within Work package 1 (WP1, Fluorescent Organic Nanoparticles, FONs), we developed a panel of advanced fluorescent nanoparticles (NPs) and characterized their optical properties.
Within Task 1.1 (Polymer FONs), we obtained the following results. (1) Our counterion-based approach for encapsulation of rhodamine dyes into polymer NPs was extended to cyanines, which allowed varying the colour of NPs from green till red (Andreiuk et al, Small, 2017). Hydrophobic counterion was crucial to prevent self-quenching of cyanine dyes inside polymer matrix and ensure formation of small particles. Mixing nanoparticles of three colours in different proportions generates a homogeneous RGB (red, green, and blue) barcode in cells, which is transmitted through many cell generations. Cell barcoding has already been validated on 7 cell lines, 13 colour codes, and it enables simultaneous tracking of co-cultured barcoded cell populations for >2 weeks. One European company plans to commercialize this technology. (2) An important problem was to find also the most optimal counterion that ensures effective encapsulation and minimize self-quenching of encapsulated dyes. In this respect we synthesized an aluminium-based fluorinated counterion F9-Al (Andreiuk et al, Mater. Chem. Front., 2017). It was found that F9-Al (a) strongly improves encapsulation efficiency of octadecyl rhodamine B dye compared to counterions used before; (b) effectively prevents aggregation-caused quenching, similarly to the best counterions used so far; (c) enables preparation of NPs that are 33-fold brighter than commercial quantum dots QD585; (d) surpasses the best counterions in stability of dye-loaded NPs against leaching in living cells. (3) In the search for fluorescent NPs exhibiting improved characteristics, we tested other polymer matrices, such as poly(methyl methacrylate) (PMMA), and polycaprolactone (PCL). In particular, we found that PMMA NPs present superior brightness and photostability in comparison to originally developed poly(lactic-co-glycolic acid) (PLGA) NPs (Reisch et al, ACS Appl. Mater. Interfaces, 2017). The obtained 35 nm NPs were nearly 100 times brighter than quantum dots. Remarkably, unlike PLGA NPs undergoing complete ON/OFF switching, PMMA and PCL NPs exhibited stable emission, which is related to different organization of dyes inside polymer matrix. This work will help us to select right polymers for developments of nanoprobes (WP2). (4) Operation in biological media requires modification of NPs with polar groups that could prevent their non-specific interactions. We showed that Pluronic block copolymer surfactant is able to bind to the surface of PLGA and PMMA NPs and thus decrease non-specific interactions of NPs with cells and improve their stability in biological media. The use of Pluronic block copolymer can be considered as complementary to covalent modification of NPs (Heimburger et al, in preparation).
Within Task 1.2 (Micellar Fluorescent Organic Nanoparticles) the following results were obtained. (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).
Within Task 1.3 (Photophysics of FONs and Collective FRET) on photophysics of NPs we made the following progress. (1) Energy transfer (FRET) was systematically studied in different micellar NPs (Shulov et al, Nanoscale, 2015, Chem. Commum, 2016, Angew. Chem. Int. Ed., 2016). We showed that single energy acceptor can produce highly efficient FRET from the whole particle. (2) In dye-loaded polymer NPs FRET was studied with particular focus on systems where large number of donors transfer energy to a single acceptor. Here, we showed that our 60 nm PMMA polymer nanoparticles containing >10,000 rhodamine-based donor dyes can efficiently transfer energy to 1–2 acceptors (Trofymchuk et al, Nature Photonics, 2017). As a result, the emission of Cy5-based acceptor dye is amplified ~1,000 and. This unprecedented amplification of the acceptor dye emission enables observation of single molecules at illumination powers that are >10,000-fold lower than typically required in single-molecule measurements. Finally, using a basic microscopy set-up, we could detect single cyanine (Cy5) molecules by simply shining divergent light on the sample at powers equivalent to sunlight. This is the key finding for the development of FRET-based nanoprobes in WP2. These results were published in Nature Photonics and the patent application was filed.

Within WP2 (Nanoprobe synthesis and evaluation), we started with Task 2.1 (FRET acceptors and superquenchers), where 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), 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) In order to obtain a powerful FRET acceptor for NPs (Task 2.1) we synthesized a dendritic octamer of squaraine dye, which showed turn-on response to environment polarity (Ashokkumar et al, in preparation). Indeed, according to photophysical studies, this squaraine octamer, exhibited poor fluorescence in water due to the folded form with π-stacked self-quenched dyes, whereas in organic solvents (dioxane), the octamer unfolds showing 335-fold fluorescence enhancement. In order to convert this octamer into turn-on probe for biomolecules (Task 2.2 Nanoprobes for membrane receptors), it was modified with cyclic RGD (cRGD) ligand for targeting integrin receptors. Our cellular studies showed that the new probe binds specifically cell surface in the receptor-specific fashion. Due to high brightness of the octamer, it was successfully used for receptor imaging and was found to be significantly brighter than single dye-based integrin probe (Ashokkumar et al, in preparation). These results are of two-fold importance: (i) we obtained an environment-sensitive building block that can be used as FRET acceptor in the nanoprobes for receptors (in Task 2.2); (ii) we obtained the first prototype of turn-on nanoprobe for integrin receptors, which addresses Task 2.2.
In Task 2.2 to target membrane receptors, we also selected 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).
Within Task 2.3 (Nanoprobes for amplified photochemical internalization), some initial studies on photo-controlled delivery into the cells have been realized on lipid NPs. We have shown that lipid-nanocarriers encapsulating large amount of dyes (Nile Red derivative) are capable to deliver their content into the cells after illumination with a visible light (Bouchaala et al, Colloids Surf. B, 2017). This work provides us insights into methods that we could use to delivery our nanoprobes into living cells.
Within Task 2.4 (Nanoprobes for nucleic acids), we have already obtained the first prototype of a FRET-based nanoprobe exhibiting high sensitivity to nucleic acids (Melnychuk et al, in preparation). Thus, we obtained the key proof-of-concept for successful realization of this Task.

Within WP3 (Cellular applications of nanoprobes), which started recently, we have already addressed in part Task 3.2 (Internalization of nanoprobes and cytotoxicity). (1) Evaluation of PLGA NPs of different colour in living cells shows that these NPs internalize by endocytosis. Due to identical endocytosis for NPs of different colour, we were able to develop a unique approach of colour coding of living cells simply by mixing particles of three colours in the cellular medium. Importantly, the labelling inside the cells preserved for >2 weeks and NPs showed almost negligible cytotoxicity. Therefore, our NPs were applied for long-term cell labeling in vitro and in vivo. The approach was validated on seven cell lines and it was applied for tracking simultaneously 6 cell lines in culture. It was also applied on zebrafish to track 6 populations of cancer cells and to study embryonic development (Andreiuk et al, Small, 2017). (2) We studied the stability of dye-loaded NPs against leakage of the encapsulated dyes: new counterions that outperform our “golden standard” counterion (F5-TPB) in terms of stability against leakage (Andreiuk et al, Mater. Chem. Front., 2017). (3) We also characterized our cross-linked micellar NPs in cell culture. We found that similarly to dye-loaded polymer NPs, they enter the cells by endocytosis. Moreover, our FRET-based studies showed that these cross-linked NPs are highly stable in cellular environment without signs on dye leakage and particle disintegration.

Finally, in addition to 15 research articles and 1 patent application, we have also published two important review articles on the subject of BrightSens project: on fluorescent polymer nanoparticles (Reisch and Klymchenko, Small, 2016) and fluorescent molecular probes (Klymchenko Accounts of Chemical Research, 2017). Both reviews are “Highly cited” articles according to ISI Web of Knowledge.
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 micellar 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 surfactant adsorption on polymer NPs surface was addressed showing that it can stabilize NPs and decrease their non-specific interactions with cells (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, Small, 2017).
6) The emission of NPs can be tuned from complete ON/OFF switching to stable emission by changing the polymer matrix of NPs (Reisch et al, ACS Appl. Mater. Interfaces, 2017).
7) We found that counterion-based approach for preparation of bright fluorescent polymer NPs can be extended to another counterion family based on fluorinated aluminium complex, showing superior properties in terms of dye encapsulation (Andreiuk et al, Mater. Chem. Front., 2017).
8) Giant light-harvesting nanoantenna was developed exhibiting unprecedented FRET efficiency from >10000 dyes to a single acceptor (Trofymchuk et al, Nature Photonics, 2017). It enabled signal amplification from a single fluorescent molecule (FRET acceptor) >1000-fold and detection of single molecules at excitation equivalent to ambient light, which has never been realized to date.

All these developments will have direct impact on the next steps of the project, notably for preparation of nanoparticle probes with amplified response to the biomolecular targets. 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 for cell imaging.
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). It was successfully applied for receptor-specific targeting of living cells.
5) We have developed the first prototype of a FRET-based nanoprobe for nucleic acids (Melnychuk et al, in preparation).

The obtained results provide important clues to development of nanoparticle-based probes for receptors and nucleic acids within BrightSens. The obtained first prototypes of fluorescent probes for receptors and nucleic acids provide the proof-of-concept for preparation of nanoprobes with exceptional sensitivity to biomolecular targets. Then, after validation in biological systems, our nanoprobes can become important tools for ultrasensitive detection of cancer markers in biological research and medical diagnostics.