Giulia Capizzani Gonçalves

Glioblastoma multiforme is one of the most aggressive primary brain tumours and remains highly resistant to conventional therapeutic approaches. Photodynamic therapy has emerged as a promising alternative. However, the clinical application of many photosensitisers is limited by low stability, poor solubility, and non-specific distribution in biological systems. In this study, iron oxide nanoparticles (SPIONs) were synthesised by a coprecipitation method and subsequently functionalised with 3-aminopropyltriethoxysilane (APTES) to enable the conjugation of the photosensitiser Chlorin E6 (Ce6). The resulting SPIONs@APTES–Ce6 nanoprobes were characterised using dynamic light scattering, Zeta Potential analysis, UV–Vis spectroscopy, fluorescence spectroscopy, and Fourier Transform infrared spectroscopy, confirming successful surface functionalisation and preservation of the photophysical properties of Ce6. The photodynamic activity of the nanoprobes was evaluated in vitro using the M059J glioblastoma cell line. Cell viability was assessed through complementary assays, including MTT, Trypan Blue exclusion, and resazurin reduction. The nanoprobes exhibited low dark cytotoxicity under the tested conditions. Upon light activation at 660 nm, a significant reduction in cell viability was observed, demonstrating effective photodynamic activity. These findings suggest that SPIONs@APTES–Ce6 nanoprobes may represent a potential nanostructured platform for further investigation in glioblastoma photodynamic therapy.

The overexpressed epidermal growth factor receptor (EGFR) in lung, breast, and colorectal cancers is associated with an increased risk of mortality. Therefore, targeted photodynamic therapy (PDT) has emerged as an alternative for cancer treatment. To enable EGFR-directed PDT, this study reports the synthesis of a bifunctional nanoprobe (NP-BF) based on gold nanoparticles (AuNPs) functionalized with the monoclonal antibody cetuximab (CX) and the photosensitizer chlorin e6 (Ce6) using carbodiimide chemistry. The resulting NP-BF showed spectroscopic evidence of successful conjugation through amide bond formation, preservation of the structural features of cetuximab, and retention of the photophysical properties of Ce6 after functionalization. In addition, the nanosystem remained colloidally stable, exhibiting a zeta potential of - 40.7 mV and an increase in hydrodynamic diameter of approximately 9 nm, consistent with antibody incorporation. A cellular uptake assay using the MDA-MB-468 cell line revealed internalization with maximum uptake in 60 min, corresponding to approximately 1.32 × 10⁵ nanoprobes per cell. PDT assays demonstrated a cytotoxic response with cell viability dropped below 10% at concentrations ≥ 1.62 μmol L^-1 (≈ 0.97 µg mL^-1 CE6 equivalent) under 660 nm irradiation, while non-irradiated samples preserved viability above 90%, confirming negligible dark toxicity. These results demonstrate the successful synthesis and functionalization of NP-BF and its effective photodynamic activity in cancer cells.

Conventional cancer treatments have limited efficacy for aggressive subtypes such as triple-negative breast cancer (TNBC), which points to the importance of new therapeutic strategies. Functionalized nanoparticles in conjunction with photodynamic therapy (PDT) represent a promising alternative. Additionally, 3D cell culture emerges as a more effective model, as it better replicates the structural and functional characteristics of the tumor microenvironment. In this study, 3D microtumors of TNBC were cultivated and treated with PDT using gold nanoparticles functionalized with Chlorin e6 (AuNPs@Ce6). Cell viability was assessed using the MTT colorimetric assay, combined with histological analysis using hematoxylin-eosin staining. The MTT assay and histological evaluation of the 3D spheroids demonstrated that PDT with AuNPs@Ce6 effectively reduced cell viability and induced necrotic morphological changes, while maintaining biocompatibility with the non-irradiated control group. These findings reinforce the potential of this approach for further investigation in TNBC models and underscore the value of 3D cultures as physiologically relevant and ethical alternatives to animal testing.