Dear Dr. Milena Mattes Cerveira

You may use fluorescent labeling. Attach a fluorophore to either the antibiotic, the metal, or a component of the nanocomplex. Then observe the labeled nanocomplexes using a fluorescence microscope, which provides high spatial resolution and can track particles in real-time or after fixation. To identify translocation, use live-cell imaging that allows for the direct visualization of nanoparticle uptake, movement, and distribution within the macrophage over time.

Alternatively, you may use nanoparticle tracking analysis (NTA) which visualizes and analyzes the movement of nanoparticles based on light scattering and Brownian motion. It can analyze fluorescently labeled nanoparticles and provide information on size, concentration, and movement over time, which can help understand translocation patterns.

1. Labelling of an antibiotic (like a protein antibiotic):

Choose a probe that is compatible with the antibiotic's functional groups. Common probes are Alexa Fluor dyes or BODIPY labels with NHS or maleimide reactive groups. Adjust the antibody's concentration and buffer conditions to a pH that promotes reaction with the chosen probe but minimizes probe decomposition. Mix the fluorescent probe with the antibiotic. The reaction typically occurs at room temperature for about an hour. For example, NHS-reactive probes react with lysine residues on proteins, while maleimide probes target cysteines. Then quickly quench the reaction by adding a neutralizing buffer. Remove unreacted probes and components using methods like dialysis or chromatography.

2. Labeling a Nanocomplex:

If the nanocomplex is polymer-based, attach fluorescent probes to the terminal end groups of the polymers before incorporating them into the nanoparticle. Formulate the nanoparticle with these pre-labeled polymers to create more stable, labeled nanoparticles; or synthesize or purchase nanoparticles that are inherently fluorescent, such as carbon quantum dots (CQDs). Encapsulate the antibiotic within these fluorescently active nanocomplexes; or use fluorescently labeled reagents, such as fluorophore-conjugated polymers or molecules, during the synthesis or assembly of the nanocomplex.

Regards,

Malcolm Nobre