A fluorescent quantum dot conjugate to probe the interaction of Enterolobium contortisiliquum trypsin inhibitor with cancer cells.

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

Multimodal highly fluorescent-magnetic nanoplatform to target transferrin receptors in cancer cells.

BACKGROUND: Site-specific multimodal nanoplatforms with fluorescent-magnetic properties have great potential for biological sciences. For this reason, we developed a multimodal nanoprobe (BNPs-Tf), by covalently conjugating an optical-magnetically active bimodal nanosystem, based on quantum dots and iron oxide nanoparticles, with the human holo-transferrin (Tf). METHODS: The Tf bioconjugation efficiency was evaluated by the fluorescence microplate assay (FMA) and the amount of Tf immobilized on BNPs was quantified by fluorescence spectroscopy. Moreover, relaxometric and fluorescent properties of the BNPs-Tf were evaluated, as well as its ability to label specifically HeLa cells. Cytotoxicity was also performed by Alamar Blue assay. RESULTS: The FMA confirmed an efficient bioconjugation and the fluorescence spectroscopy analysis indicated that 98% of Tf was immobilized on BNPs. BNPs-Tf also presented a bright fluorescence and a transversal/longitudinal relaxivities ratio (r2/r1) of 65. Importantly, the developed BNPs-Tf were able to label, efficiently and specifically, the Tf receptors in HeLa cells, as shown by fluorescence and magnetic resonance imaging assays. Moreover, this multimodal system did not cause noteworthy cytotoxicity. CONCLUSIONS: The prepared BNPs-Tf hold great promise as an effective and specific multimodal, highly fluorescent-magnetic, nanoplatform for fluorescence analyses and T2-weighted images. GENERAL SIGNIFICANCE: This study developed an attractive and versatile multimodal nanoplatform that has potential to be applied in a variety of in vitro and in vivo studies, addressing biological processes, diagnostic, and therapeutics. Moreover, this work opens new possibilities for designing other efficient multimodal nanosystems, considering other biomolecules in their composition able to provide them important functional properties.