Advances in the study of spheroids as versatile models to evaluate biological interactions of inorganic nanoparticles.

Spheroids are in vitro three-dimensional multicellular microstructures able to mimic the biological microenvironment, including the complexity of tumor architecture. Therefore, results closer to those expected for in vivo organisms can be reached using spheroids compared to the cell culture monolayer model. Inorganic nanoparticles (NPs) have also been playing relevant roles in the comprehension of biological processes. Moreover, they have been probed as novel diagnostic and therapeutical nanosystems. In this context, in this review, we present applications, published in the last five years, which show that spheroids can be versatile models to study and evaluate biological interactions involving inorganic NPs. Applications of spheroids associated with (i) basic studies to assess the penetration profile of nanostructures, (ii) the evaluation of NP toxicity, and (iii) NP-based therapeutical approaches are described. Fundamentals of spheroids and their formation methods are also included. We hope that this review can be a reference and guide future investigations related to this interesting three-dimensional biological model, favoring advances to Nanobiotechnology.

Evaluating internalization and recycling of folate receptors in breast cancer cells using quantum dots.

Folic acid (FA) regulates metabolic activities essential to the human body. FA receptor (FR) overexpression has been reported for many cancers, but there are still few or conflicting data about FRs in breast cancer cells. Quantum dots (QDs) have arisen as tools to elucidate aspects on FRs, due to their unique physicochemical properties. Herein, QDs conjugated to FA were explored to study the internalization and recycling of FRs in breast cancer cells, using HeLa as an out-group control. QDs were covalently conjugated to FA under different conditions. The best conjugate was applied to study FRs in HeLa, MCF7, MDA-MB231, and T47D cells applying confocal microscopy and flow cytometry analyses. The conjugation efficiency and specificity were evaluated, respectively, using fluorescence correlation spectroscopy (FCS) and saturation assays. FCS confirmed the effectiveness of the conjugation. HeLa and T47D had/internalized a higher amount of FRs (95% and 90% of labeling, respectively) than MDA-MB231 cells (68%). MCF7 cells seem to have very low functional FRs (3%). Saturation assays proved the specificity of QD-FA conjugates and suggested that FR recycling rate is low in the majority of cells studied, except for T47D. QD-FA conjugates were successfully developed. Therapies targeting FRs may be more effective for HeLa, T47D, and MDA-MB231.

Quantum dots functionalized with 3-mercaptophenylboronic acids as novel nanoplatforms to evaluate sialic acid content on cell membranes.

Sialic acids (SAs) modulate essential physiological and pathological conditions, including cell-cell communication, immune response, neurological disorders, and cancer. Besides, SAs confer negative charges to cell membranes, also contributing to hemorheology. Phenylboronic acids, called as mimetic lectins, have been highlighted to study SA profiles. The association of these interesting molecules with the optical properties of quantum dots (QDs) can provide a deeper/complementary understanding of mechanisms involving SAs. Herein, we explored the thiol affinity to the QD surface to develop a simple, fast and direct attachment procedure to functionalize these nanocrystals with 3-mercaptophenylboronic acids (MPBAs). The functionalization was confirmed by fluorescence correlation spectroscopy and inductively coupled plasma spectrometry. The conjugate specificity/efficiency was proved in experiments using red blood cells (RBCs). A labeling >90% was found for RBCs incubated with conjugates, which reduced to 17% after neuraminidase pretreatment. Moreover, QDs-MPBA conjugates were applied in a comparative study using acute (KG-1) and chronic (K562) myelogenous leukemia cell lines. Results indicated that KG-1 membranes have a greater level of SA, with 100% of cells labeled and a median of fluorescence intensity of ca. 2.5-fold higher when compared to K562 (94%). Therefore, this novel QDs-MPBA conjugate can be considered a promising nanoplatform to evaluate SA contents in a variety of biological systems.

Hydrophilic Quantum Dots Functionalized with Gd(III)-DO3A Monoamide Chelates as Bright and Effective T1-weighted Bimodal Nanoprobes.

Magnetic resonance imaging (MRI) is a powerful non-invasive diagnostic tool that enables distinguishing healthy from pathological tissues, with high anatomical detail. Nevertheless, MRI is quite limited in the investigation of molecular/cellular biochemical events, which can be reached by fluorescence-based techniques. Thus, we developed bimodal nanosystems consisting in hydrophilic quantum dots (QDs) directly conjugated to Gd(III)-DO3A monoamide chelates, a Gd(III)-DOTA derivative, allowing for the combination of the advantages of both MRI and fluorescence-based tools. These nanoparticulate systems can also improve MRI contrast, by increasing the local concentration of paramagnetic chelates. Transmetallation assays, optical characterization, and relaxometric analyses, showed that the developed bimodal nanoprobes have great chemical stability, bright fluorescence, and high relaxivities. Moreover, fluorescence correlation spectroscopy (FCS) analysis allowed us to distinguish nanosystems containing different amounts of chelates/QD. Also, inductively coupled plasma optical emission spectrometry (ICP - OES) indicated a conjugation yield higher than 75%. Our nanosystems showed effective longitudinal relaxivities per QD and per paramagnetic ion, at least 5 times [per Gd(III)] and 100 times (per QD) higher than the r1 for Gd(III)-DOTA chelates, suitable for T1-weighted imaging. Additionally, the bimodal nanoparticles presented negligible cytotoxicity, and efficiently labeled HeLa cells as shown by fluorescence. Thus, the developed nanosystems show potential as strategic probes for fluorescence analyses and MRI, being useful for investigating a variety of biological processes.