RESUMEN
Selenium is a component of selenoproteins, which plays a crucial role in cellular redox homeostasis, thyroid metabolism, and DNA synthesis. Selenium has pleiotropic effects like antioxidant and anti-inflammatory activities; however, excess intake of selenium can imbalance such processes. The effects of selenium on human health are numerous and complex, demanding additional research to monitor the flux rate of selenium. Here, we have created a noninvasive and highly efficient genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor, SelFS (Selenium FRET-Sensor), for real-time monitoring of selenium at the cellular and subcellular levels. The construct of the nanosensor contains a selenium-binding protein (SeBP) as the selenium-detecting element inserted between the green fluorescent protein variants enhanced cyan fluorescent protein and Venus. In the presence of selenium, SelFS brings a conformational change, which is seen in the form of FRET. In vitro studies showed that SelFS is highly specific and selective for selenium and stable at an altered pH range from 5.0 to 8.0. SelFS is a flexible and dynamic tool for the detection of selenium in both prokaryotes and eukaryotes in a noninvasive way, with a binding constant (K d) of 0.198 × 10-6 M as compared to its mutants. The developed nanosensor can provide us a reporter tool for a wide range of industrial and environmental applications, which will help us to understand its functions in biological systems.
RESUMEN
ß−sitosterol is the most abundant type of phytosterol or plant sterol and can be found in various plant dietary sources including natural oils, soy products, and nuts. Numerous studies have demonstrated the potential therapeutic and clinical applications of ß−sitosterol including lowering low-density lipoprotein and cholesterol levels, scavenging free radicals in the body, and interestingly, treating and preventing cancer. This study focuses on synthesizing and characterizing ß−sitosterol encapsulated Alginate/Chitosan nanoparticles (ß−sito−Alg/Ch/NPs) and evaluating their effectiveness in breast cancer treatment and their pharmacokinetic profile in vivo. The synthesized NPs, which incurred a mean size of 25 ± 1 nm, were extensively characterized in vitro for various parameters including surface charge and morphology. The NPs were further analyzed using DSC, FT-IR, thermogravimetry and X-ray diffraction studies. The release of ß−sito from NPs was carried out in a bio-relevant medium of pH 7.4 and pH 5.5 and samples were drawn off and analyzed under time frames of 0, 8, 16, 32, 64, 48, 80, and 96 h, and the best kinetic release model was developed after fitting drug release data into different kinetic models. The metabolic activity of MCF-7 cells treated with the prepared formulation was assessed. The radical scavenging potential of ß−sito−Alg/Ch/NPs was also studied. The pharmacokinetic parameters including Cmax, Tmax, half-life (t1/2), and bioavailability were measured for ß−sito−Alg/Ch/NPs as compared to ß−sito−suspension. The ß−sito−Alg/Ch/NPs stability was assessed at biological pH 7.4. The % drug release in PBS of pH 7.4 reportedly has shown 41 ± 6% vs. 11 ± 1% from ß−sito−Alg/Ch/NPs and ß−sito−suspension. In acidic pH 5.5 mimicking the tumor microenvironment has shown 75 ± 9% vs. 12 ± 4% drug release from ß−sito−Alg/Ch/NPs and ß−sito−suspension. When compared to the ß−sito−suspension, the ß−sito−Alg/Ch/NPs demonstrated greater cytotoxicity (p < 0.05) and ~3.41-fold higher oral bioavailability. Interestingly, this work demonstrated that ß−sito−Alg/Ch/NPs showed higher cytotoxicity due to improved bioavailability and antioxidant potential compared to the ß−sito−suspension.