KRAS Promoter G-Quadruplexes from Sequences of Different Length: A Physicochemical Study

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5′-AGGGCGGTGTGGGAATAGGGAA-3′ (KRAS 22RT) and 5′-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3′ (KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs

Host–guest inclusion complex of quercetin and hydroxypropyl-β-cyclodextrin

Quercetin (QCT), a flavonoid derived from many fruits and vegetables, is endowed with manifold biological properties, such as the ability to elicit a strong inhibitory effect on the growth of several tumor cell lines. Unfortunately, the pharmacological application of QCT is severely restricted by its inherent hydrophobicity and consequent low in vivo bioavailability. The therapeutic potential of QCT can be unraveled by enhancing its solubility through the formation of a host–guest complex with hydroxypropyl-β-cyclodextrin (HPβCD). In this study, HPβCD·QCT complex has been obtained in liquid phase, at 37 °C and under a prolonged mixing (72 h), and using two buffers at pH = 3.6 and pH = 8.0. Phase solubility and differential scanning calorimetry (DSC) studies revealed that, at pH = 8.0, the complex was obtained with a 1:1 stoichiometric ratio and a strong enhancement of QCT solubility, while in acidic buffer complex formation was significantly thwarted. The affinity constant was calculated by isothermal calorimetry at pH = 8 and was found to be 489 ± 38 M−1, in good agreement with the value indirectly obtained from phase solubility tests 394 ± 101 M−1. The results confirmed the formation of the inclusion complex between QCT and HPβCD and highlight the importance of the choice of the appropriate solvent, pH, temperature and mixing time on the formation of host guest inclusion complex with active ingredient(s) and HPβCD. © 2017 Akadémiai Kiadó, Budapest, Hungar

Physicochemical and in vitro biological validation of food grade secondary oil in water nanoemulsions with enhanced mucus-adhesion properties

Among oral delivery systems, oil in water nano-emulsions (O/W NEs) are of particular interest to improve pharmacokinetics of lipophilic compounds. Recently, we have implemented a successful strategy to improve O/ W NEs stability, based on a polymeric coating on an oil core, namely secondary O/W NEs, through the use of pharma grade formulations. However, in the field of food supplements, food grade materials are the top choice since they combine safety and cost effectiveness. Here, we have replaced pharma grade (PG) with food grade (FG) materials in the preparation of the polymer coated O/W NEs, and performed a comparative study between the two formulations to assess the FG one. At the same time, in order to provide formulations with enhanced mucus-adhesion to the intestinal barrier, secondary O/W NEs were prepared by adding thiol groups to chitosan (Ct) via a simple non-covalent procedure based on N-acetyl-cysteine (NAC) salification, thus easily implementable to a food supplement formulation. PG and FG formulations, in different materials combinations, were prepared and physico-chemically characterized (DLS, 1H NMR, ITC, CRYO-TEM) showing similar behaviour. FG formulations (NEs, Ct-NEs and Ct-NAC-NEs) loaded with curcumin were prepared and compared with the free drug in terms of drug bioaccessibility through the INFOGEST protocol confirming improved bioaccessibility. Very interestingly, by comparing mucus-adhesion properties of the two polymeric coatings (Ct and Ct-NAC) within an intestine on chip device able to mimic the complex intestinal functions, a significant enhancement in the mucus-adhesive properties of the proposed novel Ct-NAC-NE formulation was observed with respect to Ct due to the presence of thiol groups. Nonetheless, in-vivo assays are required as a final assessment of the proposed system