Over the years, the scientific importance of nanoparticles for biomedical applications has increased. The high stability and biocompatibility, together with the low toxicity of the nanoparticles developed lead to their use as targeted drug delivery systems, bioimaging systems, and biosensors. The wide range of nanoparticles size, from 10 nm to 1 μm, as well as their optical properties, allow them to be studied using microscopy and spectroscopy techniques. In order to be effectively used, the physicochemical properties of nanoparticle formulations need to be taken into account, namely, particle size, surface charge distribution, surface derivatization and/or loading capacity, and related interactions. These properties need to be optimized considering the final nanoparticle intended biodistribution and target. In this review, we cover light scattering based techniques, namely dynamic light scattering and zeta-potential, used for the physicochemical characterization of nanoparticles. Dynamic light scattering is used to measure nanoparticles size, but also to evaluate their stability over time in suspension, at different pH and temperature conditions. Zeta-potential is used to characterize nanoparticles surface charge, obtaining information about their stability and surface interaction with other molecules. In this review, we focus on nanoparticle characterization and application in infection, cancer and cardiovascular diseases.

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15–34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15–34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90–120 and 5–30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15–34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15–34) binds to and disrupts lipid membranes and also observed that Ctn(15–34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15–34), suggesting Ctn(15–34) as a promising lead for development as an antibacterial/antitumor agent.

Many cellular phenomena occur on the biomembranes. There are plenty of molecules (natural or xenobiotics) that interact directly or partially with the cell membrane. Biomolecules, such as several peptides (e.g., antimicrobial peptides) and proteins, exert their effects at the cell membrane level. This feature makes necessary investigating their interactions with lipids to clarify their mechanisms of action and side effects necessary. The determination of molecular lipid/water partition constants (K _p ) is frequently used to quantify the extension of the interaction. The determination of this parameter has been achieved by using different methodologies, such as UV-Vis absorption spectrophotometry, fluorescence spectroscopy and ζ-potential measurements. In this work, we derived and tested a mathematical model to determine the K _p from ζ-potential data. The values obtained with this method were compared with those obtained by fluorescence spectroscopy, which is a regular technique used to quantify the interaction of intrinsically fluorescent peptides with selected biomembrane model systems. Two antimicrobial peptides (BP100 and pepR) were evaluated by this new method. The results obtained by this new methodology show that ζ-potential is a powerful technique to quantify peptide/lipid interactions of a wide variety of charged molecules, overcoming some of the limitations inherent to other techniques, such as the need for fluorescent labeling.

Mammalian cell membranes regulate homeostasis, protein activity and cell signaling. The charge at the membrane surface has been correlated with these key events. Although mammalian cells are known to be slightly anionic, quantitative information on the membrane charge and the importance of electrostatic interactions in pharmacokinetics and pharmacodynamics remain elusive. Recently, we reported for the first time that brain endothelial cells are more negatively charged than human umbilical cord cells, using zeta-potential dynamic light scattering. Here, we hypothesize that anionicity is a key feature of the blood-brain barrier and contributes to select which compounds cross into the brain. For the sake of comparison, we also studied the membrane surface charge of blood components – red blood cells, platelets and peripheral blood mononuclear cells. To further quantitatively correlate the negative zeta-potential values with membrane charge density, model membranes with different percentages of anionic lipids were also evaluated. From all the cells tested, brain cell membranes are the most anionic and the ones having their lipids mostly exposed, which explains why lipophilic cationic compounds are more prone to cross the blood-brain barrier.