Guanidine-to-piperidine switch affords high affinity small molecule NPFF ligands with preference for NPFF1-R and NPFF2-R subtypes.

The Neuropeptide FF (NPFF) receptor system is known to modulate opioid actions and has been shown to mediate opioid-induced hyperalgesia and tolerance. The lack of subtype selective small molecule compounds has hampered further exploration of the pharmacology of this receptor system. The vast majority of available NPFF ligands possess a highly basic guanidine group, including our lead small molecule, MES304. Despite providing strong receptor binding, the guanidine group presents a potential pharmacokinetic liability for in vivo pharmacological tool development. Through structure-activity relationship exploration, we were able to modify our lead molecule MES304 to arrive at guanidine-free NPFF ligands. The novel piperidine analogues 8b and 16a are among the few non-guanidine based NPFF ligands known in literature. Both compounds displayed nanomolar NPFF-R binding affinity approaching that of the parent molecule. Moreover, while MES304 was non-subtype selective, these two analogues presented new starting points for subtype selective scaffolds, whereby 8b displayed a 15-fold preference for NPFF1-R, and 16a demonstrated an 8-fold preference for NPFF2-R. Both analogues showed no agonist activity on either receptor subtype in the in vitro functional activity assay, while 8b displayed antagonistic properties at NPFF1-R. The calculated physicochemical properties of 8b and 16a were also shown to be more favorable for in vivo tool design. These results indicate the possibility of developing potent, subtype selective NPFF ligands devoid of a guanidine functionality.

CdTe-GSH as luminescent biomarker for labeling the larvicidal action of WSMoL lectin in Aedes aegypti larvae.

Mosquito-borne arboviruses compromise human health worldwide. Due to resistance to chemical insecticides, natural compounds have been studied to combat mosquitoes. Previous works have demonstrated a larvicidal activity of the water-soluble Moringa oleifera lectin (WSMoL) against Aedes aegypti, suggesting a mechanism of action based on the interaction between lectin and chitin present in the larvae's peritrophic matrix. In this work, it was investigated the WSMoL activity against Aedes aegypti larvae, by using luminescent bioconjugates of WSMoL conjugated to l-glutathione capped CdTe quantum dots. The conjugation was confirmed by ITC experiments, presenting high enthalpy associated to hydrogen bond interactions between nanoparticles and lectins. The bioconjugate luminescence stability was evaluated by the quantum yield (QY) at different pHs, ionic strengths and heat treatment time. The best parameters reached were pH 7.0, absence of electrolytes and heat treatment, giving QY = 4.4 %. The larvae were exposed to the bioconjugates and analyzed by confocal and fluorescence microscopy. CdTe-WSMol were detected along the entire midgut tract, suggesting a strong interaction with peritrophic matrix and lumen of the Aedes aegypti.

Studies on intracellular delivery of carboxyl-coated CdTe quantum dots mediated by fusogenic liposomes.

The use of Quantum Dots (QDs) as fluorescent probes for understanding biological functions has emerged as an advantageous alternative over application of conventional fluorescent dyes. Intracellular delivery of QDs is currently a specific field of research. When QDs are tracking a specific target in live cells, they are mostly applied for extracellular membrane labeling. In order to study intracellular molecules and structures it is necessary to deliver free QDs into the cell cytosol. In this work, we adapted the freeze and thaw method to encapsulate water dispersed carboxyl-coated CdTe QDs into liposomes of different compositions, including cationic liposomes with fusogenic properties. We showed that labeled liposomes were able to fuse with live human stem cells and red blood cells in an endocytic-independent way. We followed the interactions of liposomes containing QDs with the cells. The results were minutely discussed and showed that QDs were delivered, but they were not freely diffused in the cytosol of those cells. We believe that this approach has the potential to be applied as a general route for encapsulation and delivery of any membrane-impermeant material into living cells.

Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells.

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals - Quantum Dots (QDs) - have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties - wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.