Results 2018: 

The research activities in stage I (2018) of the project were carried out according to the plan of implementation, leading to the achievement of all the objectives. At the beginning of the project, the partners have defined their work strategy concerning chemical synthesis of molecules, fabrication of fluorescent CA in the NIR, selection of biological systems and protocols.

► Partner P2 has conducted a series of molecular modeling studies and retrosynthetic analyzes to identify the most advantageous strategy to synthesize methyl blue analogs which exhibit the absorption band shifted from the visible to the NIR domain

► The partner CO has started procedures for the preparation of polymeric nanoparticles and polyelectrolyte (micro)capsules and performed the spectroscopic characterization of four NIR fluorophores (ICG, MB, IR780, IR820),  among them ICG was investigated as free and loaded in microcapsules.

► The Partner P1 established the biological systems and protocols for the microscopic assessment of the internalization / incorporation by cells and ovarian tumors of NIR fluorescent contrast agents fabricated by CO and P2.

Results 2019: 

The research activities in stage II (2019) of the project were carried out according to the plan of implementation, leading to the achievement of all the objectives. Specifically, in the second stage of the project, the partners performed the activities related to the chemical synthesis of novel NIR fluorescent dyes for FRα-targeting, fabrication of fluorescent CA in the NIR, investigation of the expression of FRα in a set of ovarian cell lines sensitive and resistant to cisplatin, evaluation of the in vitro toxicity of CA on different cisplatin sensitive/resistant ovarian cell lines, and uptake and intracellular internalization studies. 

► The Partner P2 synthesized a series of new phenothiazine-cored NIR fluorescent dyes containing the various functionalized phenothiazine nucleus (cyanine, porphyrin and phenothiazinium dyes, respectively). P2 also synthesized a novel phenazationium NIR fluorescent dye (G1) which was selected for validation as a contrast agent in the project. In addition, some of the synthesized phenothiazine dyes (e.g. G1) have been conjugated with folic acid for targeting the folate alpha receptor (FRα). Also, P2 continued the molecular modelling studies using the DFT method.  

► The Partner CO developed and characterized 3 classes of CA that are structurally presented as nanocontainers (micelles, protein nano-aggregates and hybrid nanoparticles, respectively) encapsulated with NIR fluorophores. Specifically, the following classes of CA have been fabricated and characterized: (1) Pluronic-F127 micelles encapsulated with IR780 iodide commercial dye (Plu-IR780) with fluorescence emission at 806 nm, and Pluronic-F127 micelles encapsulated with the novel fluorescent dye G1 (Plu-G1) synthesized by P2, with fluorescence emission at 704 nm; (2) Protein nano-aggregates encapsulated with the FDA approved indocyanine green dye (ICG) with fluorescence emission at 814 nm; and (3) multilayer hybrid polymer nanoparticles built on CaCO3 core and encapsulated with ICG. The first two classes have been functionalized with a recognition element (folic acid- FA) for targeting the NIH:OVCAR3 ovarian cancer cells. The fabricated CA (with and without FA) have been validated in cell cultures by several biological tests, confocal Raman imaging and fluorescence microscopy studies.

► The Partner P1 investigated the expression of FRα in a set of ovarian cell lines sensitive and resistant to cisplatin (NIH:OVCAR3, A2780 and A2780 cis) and found that the NIH:OVCAR3 line is characterized by the strongest expression of FOLR1 (19% of positive cells), followed by A2780 (15%) and A2780 cis (0%). P1 also evaluated the in vitro toxicity of both, the novel phenothiazine-cored NIR fluorescent dyes synthesized by P2 and the CA fabricated by CO. Dose and cellular type-dependent G1-induced cellular damage have been observed. No toxicity was observed in the case of the protein nano-aggregates encapsulated with the FDA approved ICG, even at high concentrations of ICG. IR780 free caused cell death at high concentrations. However, Plu-IR780 micelles were non-toxic at an IR780 concentration below 46 ng /ml.  

► Partner P2 continued and optimized the synthesis of new phenothiazine-cored NIR fluorescent dyes and obtained 3 new dyes through the functionalization of phenazationium tetraiodide with amino-alcools. Also, P2 synthesized new meso-phenothiazinyl-porphyrin (MFP) dyes containing acetylene units grafted at the border of the chromophore system (ethinyl-MFP 1-8), through mixed condensation of phenothiazyne-3-carbaldehyde and pyrrole benzaldehyde derivatives. The folate esters obtained after the direct esterification of phenazationium dyes with FA exhibited moderate quantum yield (43-60%) and fluorescence emission at 698 nm. Also, P2 continued the molecular modelling studies using the DFT method in order to reveal the absorption properties of the new 4 synthesized cationic (phenothiazinyl)vinyl-pyridinium dyes (FVP) and in collaboration with CO, tested the performance of the FVP1-4 dyes in cellular imaging showing green fluorescence emission inside ovarian cells.

► The partner P1 has studied the cellular internalization capability as well as the molecular effects induced by the polymeric/protein nanoparticles loaded with NIR fluorophores fabricated by CO and the dyes synthesized by P2 for three cellular lines of ovarian cancer, NIH:OVCAR3, A2780 and A2780 cis (resistant to cisplatin), respectively. Moreover, murine solid tumours models (xenograft) were obtained to be used in the next phase of the project for the evaluation of the intratumoral internalization of the contrast agents. For the selection of the patients diagnosed with ovarian cancer, P1 has investigated the tissular expression of 5 biomarkers assigned to the FOLR1 tumour target antigen to obtain an accurate characterisation of the malign therapy-resistant phenotype. Thus, the expression of proteins involved in different molecular mechanisms modulated by FOLR1 were studied, as follows: i) CAV-1 and CDH1, which are involved in the cellular progression and proliferation, respectively the increase of the tumoral cell migration capacity through the activation of the epithelial mesenchymal transition (EMT); ii) β-catenin (CTNNB1), an indicator of CAV-1; iii) CD9 and CD151 transmembrane proteins, given the role of the angiogenesis in the development of the invasive phenotype. These proteins were studied for 3 groups of tissular samples including the benign, borderline, and malign ovarian pathology. In 2020,  P1 has implemented in the surgery room the Surgical Fluorescence Microscope Zeiss Pentero acquisitioned during the prior stages of the project (see representative images below).

Results 2021:

The research activities in stage IV (2021) of the project were carried out continuously in the 01.01.2021 - 31.12.2021 period, leading to the achievement of all planned objectives.

► The project objectives related to the biological effects of the newly developed contrast agents (CA) with emission in the NIR domain and targeted binding to the folate receptor alpha (FRα) were achieved by P1 in collaboration with CO and P2. The biocompatibility and bioavailability of the newly developed CA based on Human Serum Albumin (HSA) nanoparticles loaded with Zn(II)-2,9,16,23-tetranitrophthalocyanine (phthaloNO2) fluorophore and functionalized with anti-FRα antibodies (AB), hereinafter denoted as HSA&phthaloNO2-AB NPs, have been investigated in vitro on FRα-positive ovarian cancer cell lines. The investigations revealed the cytotoxic effects induced by the intracellular internalization of CA in combination with exposure to excitation sources in resonance with the CA, predominantly through apoptosis, and a decrease in the expression of some growth factors (VEGF-A, FGF-2) and transcription factors (NFkB). In vivo investigations on mice bearing FRα-positive ovarian tumours after treatment with HSA&phthaloNO2-AB NPs proved the ability of these NPs to be taken up by tumors and, in combination with irradiation, to induce a strong cytotoxic response. The translation of the study to clinical practice aimed to evaluate the expression of FRα in the serum and tissues from patients with advanced ovarian cancer. FRα levels in serum were significantly elevated preoperatively in patients with ovarian cancer, especially in patients with advanced stages of cancer, levels that decreased postoperatively and after neoadjuvant chemotherapy. A significant correlation was observed between the FRα expression in serum and tissue. Immunohistochemical quantification of FRα expression in tissue was performed by I-score (intensity of staining) and H-score (I-score × percentage of positive cells), obtaining an average H-score of 118 for pre-chemotherapy patients, and 126 for post-chemotherapy patients. However, there were no significant changes in the H-score after chemotherapy in the pair trials, indicaing the invariability of this biomarker to the clinical features.

► The partner CO continued the research started in stage III (2020) on photoluminescent nanomaterials (gold nanoclusters, graphene oxide (GO) nanosheets and gold nanoparticles labelled with fluorophores) having as main objective the optimization of their photoluminescent properties. In particular, gold nanoclusters maintain their photoluminescence through lyophilization and redispersion in artificial tissue mimicking phantoms, while by binding GO to the PVP polymer (GO-PVP), the emission quantum yield of GO improves. Moreover, a promising result was obtained in the case of the contrast agents obtained via metal enhanced fluorescence by employing denaturated BSA protein as an agent for binding and positioning of the ICG fluorophore on the metal surface. The first two types of photoluminescent nanomaterials were evaluated inside tissue mimicking phantoms using standard confocal microscopy techniques (FLIM, Raman) and double-scan confocal microscopy (RCM), respectively, and their potential as contrast agents was demonstrated. In 2021, the CO partner broadened the capabilities of the RCM Confocal Fluorescence Microscope in the NIR region acquired in stage III (2020), by installing two new laser diodes and mounting the system on an active pneumatic anti-vibration table.

► Partner P2 continued the research activities started in the previous stages of the project and synthesized 12 new phenothiazinium dyes, derivatives of 5-phenothiazinil-metilidenbarbituric acid, with fluorescence emission in the 612 – 778 nm region. Also, P2 prepared new samples of folic acid esters with phenothiazine chromophore units. The fluorescence properties of the synthesized dyes were investigated in collaboration with CO using time-resolved fluorescence spectroscopy and FLIM measurements. In parallel, CO and P1 performed in vitro experiments on ovarian tumoral cell cultures and evaluated the internalization and cytotoxicity of Zn(II)-2,9,16,23-tetranitrophthalocyanine (phthaloNO2) dye synthesized by P2 in the previous stages of the project by P2.

Results 2022:

The research activities in stage V (2022) of the project were carried out continuously in the 01.01.2022 - 09.10.2022 period, leading to the achievement of all planned objectives.

► In the current stage partner P1 focused on in vitro and in vivo testing activities regarding both the cytotoxicity and the internalization in ovarian carcinoma cells of the new contrast agent synthesized by partners P2 and CO. Several animal models have been developed to perform in vivo experiments: the human ovarian xenograft model with subcutaneous injection of OVCAR3 cells in SCID Beige nude mice, the Ehrich ascites model in CD1 immunocompetent mice, and an intraperitoneal metastasis model obtained by inoculation of B161 cells in Bl57 mice. This latter model proved to be a quick solution for obtaining peritoneal determinations with FOLR1-positive cells. The animal experiments had two directions: [1] an ex vivo model in which the internalization, accumulation and retention of selected CA (free ICG, HSA&ICG NPs, FA-HSA&ICG NPs) were evaluated at tissue level at different time intervals (2, 4 hours) at the level of organs (liver, spleen) and at the level of tumor metastases; [2] in vivo observation model using the ZEISS OPMI PENTERO surgical microscope with filter for the visualization of the route of intravenously administered contrast agents in mice with xenografts and in mice with peritoneal metastases, showing a slightly increased persistence in the circulation but also in tumor level of the newly synthesized CA compared to the agent already established for clinical use, the ICG.

► The partner CO extended the research started in the previous stages on the synthesis of luminescent nanomaterials (gold nanoclusters) and the evaluation of their performance in tissue-like platforms, the so-called "phantoms". Thus, a new class of gold nanoclusters stabilized with glutathione was synthesized and characterized, interstingly with dual emission in the red (610 nm)  and NIR domain (800 nm). The preservation of their luminescence in phantoms was also demonstrated by combined FLIM-RCM studies. In addition, previously started studies on phantoms doped with BSA-AuNC were completed in terms of optimizing the concentration of BSA-AuNC inside the phantom and the excitation wavelength. Also, CO continued research on the luminescence of oxidized graphene nanosheets (GO) and GO passivated with polyvinylpyrrolidone polymer (PVP) by testing their ability to act as fluorescent contrast agents in solid phantoms, not only on the surface of the phantoms (level 2D), but also in their depth (3D level). The demonstration was made through the non-invasive re-scanning confocal microscopy using the RCM NIR Fluorescence Microscope equipment purchased in the 2020 stage of the project. The results highlight the superior performance of GO-PVP compared to free GO as fluorescent contrast agents in solid phantoms, both at 2D and 3D levels. In addition, CO investigated by means of FLIM and RCM techniques the spatial distribution and lifetime mapping of CA HSA&ICG NPs in Ehrlich ascites and mouse liver, as well as the labeling ability of human ovarian cancer tissues by FA-HSA&ICG NPs and Plu-IR780 compounds.

► During this stage, partner P2 continued the activities regarding the synthesis and structural characterization by high-resolution spectroscopic methods (NMR spectroscopy, FT-IR, UV-vis, mass spectrometry) of new phenothiazine derivatives with NIR fruorescence. Thus, new organic compounds such as cationic dyes with phenazathionium chromophore symmetrically substituted with aminoalcohols or aromatic amines and dyes with the polyheterocyclic structure of the Triad Ferrocene-Phenothiazine-Porphyrin type were obtained. The dyes present fluorescence emission with satisfactory quantum yield in the first NIR window relevant for biomedical applicability (λem = 715-650 nm). Samples of dyes were provided to partner P1 for  imaging experiments (FLIM with excitation at one and two photons) and respectively to partner CO for evaluating their cytotoxicity and ability of staining the tumor cells. Folic acid esters were prepared using cationic dyes with a symmetrically substituted phenazathionium chromophore system with amino auxochromic groups originating from aminoalcohols, these folates presenting as a characteristic a fluorescence emission with maxima located in the first NIR window.