Monoclonal Antibody-Targeted Fluorescein-5-isothiocyanate-Labeled Biomimetic Nanoapatites: A Promising Fluorescent Probe for Imaging Applications

Abstract

Multifunctional biomimetic nanoparticles (NPs) are acquiring increasing interest as carriers in medicine and basic research since they can efficiently combine labels for subsequent tracking, moieties for specific cell targeting, and bioactive molecules, e.g., drugs. In particular, because of their easy synthesis, low cost, good biocompatibility, high resorbability, easy surface functionalization, and pH-dependent solubility, nanocrystalline apatites are promising candidates as nanocarriers. This work describes the synthesis and characterization of bioinspired apatite nanoparticles to be used as fluorescent nanocarriers targeted against the Met/hepatocyte growth factor receptor, which is considered a tumor associated cell surface marker of many cancers. To this aim the nanoparticles have been labeled with Fluorescein-5-isothiocyanate (FITC) by simple isothermal adsorption, in the absence of organic, possibly toxic, molecules, and then functionalized with a monoclonal antibody (mAb) directed against such a receptor. Direct labeling of the nanoparticles allowed tracking the moieties with spatiotemporal resolution and thus following their interaction with cells, expressing or not the targeted receptor, as well as their fate in vitro. Cytofluorometry and confocal microscopy experiments showed that the functionalized nanocarriers, which emitted a strong fluorescent signal, were rapidly and specifically internalized in cells expressing the receptor. Indeed, we found that, once inside the cells expressing the receptor, mAb-functionalized FITC nanoparticles partially dissociated in their two components, with some mAbs being recycled to the cell surface and the FITC-labeled nanoparticles remaining in the cytosol. This work thus shows that FITC-labeled nanoapatites are very promising probes for targeted cell imaging applications.

Functionalized Biomimetic Magnetic Nanoparticles as Effective Nanocarriers for Targeted Chemotherapy

“…All together, these data show that, under these conditions, the specificity of the mAb‐mediated interaction is preserved up to 1 h and suggest that only after 2 h incubation some aspecific interaction of BMNPs occurs. These findings are in line with previously reported results and show that these nanoassemblies can mediate active targeting. The decrease of specificity with time agrees with previously reported results, showing that NPs of different composition can interact and possibly be internalized within cells, even in the absence of a receptor specifically recognized by a cognate ligand present on the NPs .…”

“…Cytocompatibility and Cytotoxicity of the BMNPs : Cells (≈12 × 10 3 GTL‐16 per well or 6 × 10 3 Huh7 per well) were incubated in 96‐well plates for 24 h; then different amounts of the BMNPs and nanoassemblies were added, as well as equimolar amounts of soluble DOXO. Cells were then incubated for 3 days, after which their viability was evaluated by the MTT colorimetric assay, as already described . Briefly, MTT solution (5 mg mL −1 in phosphate buffered saline (PBS) solution) was added to the plate, incubated at 37 °C for 2 h and supernatants were aspirated.…”

“…Cellular Interactions of the BMNPs : Cells (≈24 × 10 3 GTL‐16 per well or 12 × 10 3 Huh7 per well) were seeded on glass coverslips and, after 24 h, soluble DO‐24 mAb (positive control), soluble DOXO, and suspensions of the different BMNPs (presaturated in 0.4% bovine serum albumin (BSA) at 37 °C for 2 h) were added. After incubation at 37 °C for different times (from 5 min to 1 h), coverslips were washed with PBS, fixed with paraformaldehyde (PFA, 2 wt% in PBS), washed and permeabilized with tris‐buffered saline‐5% BSA, 0.1% Triton‐X100 and 5% got serum, and stained as described . Cytoskeletal actin was stained with TRITC‐phalloidin (1/200, Sigma‐Aldrich, excitation at 543 nm; emission at 560–620 nm), or FITC‐phalloidin (Sigma‐Aldrich, excitation at 488 nm; emission at 500–535 nm), mAbs with FITC‐labeled rabbit‐anti‐mouse IgG (1/100, Abcam excitation at 488 nm; emission at 500–535 nm), and nuclei with TO‐PRO‐3 (1/70, Life Technologies; excitation at 633 nm; emission at 650–750 nm).…”

Nanoformulation Design Including MamC-Mediated Biomimetic Nanoparticles Allows the Simultaneous Application of Targeted Drug Delivery and Magnetic Hyperthermia

“…Identical behaviour was shown when these BM were functionalized with the monoclonal antibody DO-24 [ 22 ]. This is the great advantage of these BMs, which do not need further coating with other molecules that could alter the nanoparticles or their performance in biological systems, as previously shown by the covering of nanoparticles with AMPTES [ 31 ]. The coupling of DOXO and of the antibody to the BMs was extensively characterized by Peigneux et al [ 22 ] (both the kinetics and thermodynamics of the coupling process and the structure and functionality of the relevant molecules after coupling), which shows that, under the conditions of the experiment, ~50% of the initial mAb in solution were first adsorbed on the BM and, after further coupling with DOXO, ~80% of the initial DOXO in solution was adsorbed at the plateau.…”

Eu-Doped Citrate-Coated Carbonated Apatite Luminescent Nanoprobes for Drug Delivery

“…For the former, biosensing and bioimaging can be approached by the use of luminescent nanoparticles that can provide the required fluorescent contrast [4]. Fluorescent inorganic and organic nanoparticles including quantum dots, silica, gold, conjugated polymers, organic dyes and structures labeled with organic dyes and monoclonal antibodies have been largely investigated [5][6][7][8][9][10][11]. Among them, and in the context of bioimaging, some organic fluorescent dyes-based nanomaterials have been reported to present favorable characteristics compared to those of inorganic nanoparticles such as higher biodegradability, biocompatibility and lower toxicity [12], especially when compared to quantum dots, which are somehow cytotoxic and show photoblinking [5,13].…”