The interaction of temozolomide (TMZ) (the main chemotherapeutic agent for brain tumors) with blood components has not been studied at the molecular level to date, even though such information is essential in the design of dosage forms for optimal therapy. This work explores the binding of TMZ to human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP), as well as to blood cell-mimicking membrane systems. Absorption and fluorescence experiments with model membranes indicate that TMZ does not penetrate into the lipid bilayer, but binds to the membrane surface with very low affinity. Fluorescence experiments performed with the plasma proteins suggest that in human plasma, most of the bound TMZ is attached to HSA rather than to AGP. This interaction is moderate and likely mediated by hydrogen-bonding and hydrophobic forces, which increase the hydrolytic stability of the drug. These experiments are supported by docking and molecular dynamics simulations, which reveal that TMZ is mainly inserted in the subdomain IIA of HSA, establishing π-stacking interactions with the tryptophan residue. Considering the overexpression of albumin receptors in tumor cells, our results propose that part of the administered TMZ may reach its target bound to plasma albumin and suggest that HSA-based nanocarriers are suitable candidates for designing biomimetic delivery systems that selectively transport TMZ to tumor cells.

This paper explores the interaction mechanism between the conjugated polyelectrolyte {[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]fluorene-phenylene}bromide (HTMA-PFP) and model lipid membranes. The study was carried out using different biophysical techniques, mainly fluorescence spectroscopy and microscopy. Results show that despite the preferential interaction of HTMA-PFP with anionic lipids, HTMA-PFP shows affinity for zwitterionic lipids; although the interaction mechanism is different as well as HTMA-PFP’s final membrane location. Whilst the polyelectrolyte is embedded within the lipid bilayer in the anionic membrane, it remains close to the surface, forming aggregates that are sensitive to the physical state of the lipid bilayer in the zwitterionic system. The different interaction mechanism is reflected in the polyelectrolyte fluorescence spectrum, since the maximum shifts to longer wavelengths in the zwitterionic system. The intrinsic fluorescence of HTMA-PFP was used to visualize the interaction between polymer and vesicles via fluorescence microscopy, thanks to its high quantum yield and photostability. This technique allows the selectivity of the polyelectrolyte and higher affinity for anionic membranes to be observed. The results confirmed the appropriateness of using HTMA-PFP as a membrane fluorescent marker and suggest that, given its different behaviour towards anionic and zwitterionic membranes, HTMA-PFP could be used for selective recognition and imaging of bacteria over mammalian cells.

Here, we present a study on the incorporation and characterization of the enzyme alkaline phosphatase (ALP) into a three-dimensional polymeric network through a green protocol to obtain transparent hydrogels (ALP@AETA) that can be stored at room temperature and potentially used as a disposable biosensor platform for the rapid detection of ALP inhibitors. For this purpose, different strategies for the immobilization of ALP in the hydrogel were examined and the properties of the new material, compared to the hydrogel in the absence of enzyme, were studied. The conformation and stability of the immobilized enzyme were characterized by monitoring the changes in its intrinsic fluorescence as a function of temperature, in order to study the unfolding/folding process inside the hydrogel, inherently related to the enzyme activity. The results show that the immobilized enzyme retains its activity, slightly increases its thermal stability and can be stored as a xerogel at room temperature without losing its properties. A small portion of a few millimeters of ALP@AETA xerogel was sufficient to perform enzymatic activity inhibition assays, so as a proof of concept, the device was tested as a portable optical biosensor for the detection of phosphate in water with satisfactory results. Given the good stability of the ALP@AETA xerogel and the interesting applications of ALP, not only in the environmental field but also as a therapeutic enzyme, we believe that this study could be of great use for the development of new devices for sensing and protein delivery.

This work describes the development and characterization of fluorescent nanocomposite hydrogels, with high swelling and absorption capacity, and prepared using a green protocol. These fluorescent materials are obtained by incorporating, for the first time, polyfluorenes-based nanoparticles with different emission bands—poly[9,9-dioctylfluorenyl-2,7-diyl] (PFO) and poly[(9,9-di-n-octylfluorenyl-2,7-diyl)-alt-(1,4-benzo-{2,1,3}-thiadiazole)] (F8BT)—into a three-dimensional polymeric network based on polyacrylamide. To this end, two strategies were explored: incorporation of the nanoparticles during the polymerization process (in situ) and embedment after the hydrogel formation (ex situ). The results show that the combination of PFO nanoparticles introduced by the ex situ method provided materials with good storage stability, homogeneity and reproducibility properties, allowing their preservation in the form of xerogel. The fluorescent nanocomposite hydrogels have been tested as a transportable and user-friendly sensing platform. In particular, the ability of these materials to specifically detect the enzyme alkaline phosphatase (ALP) has been evaluated as a proof-of-concept. The sensor was able to quantify the presence of the enzyme in an aqueous sample with a response time of 10 min and LOD of 21 nM. Given these results, we consider that this device shows great potential for quantifying physiological ALP levels as well as enzyme activity in environmental samples.

Eutectogels are advanced gel-based systems that integrate deep eutectic solvents (DES) into polymer networks. In this study, we report the first detailed characterization of an enzyme-containing eutectogel, representing a significant step toward advanced biosensing and biocatalytic applications. Specifically, we have incorporated pancreatic lipase, one of the main target enzymes in the treatment of obesity, in eutectogels via UV-induced radical polymerization of suitable precursors in appropriate DESs. Prior to immobilization, the enzyme was solubilized in selected DESs and its activity and conformational stability were evaluated using colorimetry and intrinsic fluorescence. Combinations of choline chloride/glycerol and tetramethylammonium chloride/glycerol were shown to be effective media for preserving and enhancing enzymatic function and conformational stability. The enzyme immersed in eutectogel exhibited high structural integrity and excellent thermal stability, maintaining its activity over several weeks. The ability of this new material to screen enzyme inhibitors was assessed using orlistat, a well-established anti-obesity agent. The results demonstrated clear detection of the drug’s inhibitory effect, even at nanomolar concentrations, highlighting the material’s potential as a screening platform for novel inhibitors with prospective anti-obesity activity. Furthermore, the device proved effective in quantifying drug presence, offering a promising and highly sensitive tool for pharmaceutical quality control applications.

The nanostructuration of solid matrices with lipid nanoparticles containing membrane proteins is a promising tool for the development of high-throughput screening devices. Here, sol-gel silica-derived nanocomposites loaded with liposome-reconstituted KcsA, a prokaryotic potassium channel, have been synthesized. The conformational and functional stability of these lipid nanoparticles before and after sol-gel immobilization have been characterized by using dynamic light scattering, and steady-state and time-resolved fluorescence spectroscopy methods. The lipid-reconstituted KcsA channel entrapped in the sol-gel matrix retained the conformational and stability changes induced by the presence of blocking or permeant cations in the buffer (associated with the conformation of the selectivity filter) or by a drop in the pH (associated with the opening of the activation gate of the protein). Hence, these results indicate that this novel device has the potential to be used as a screening platform to test new modulating drugs of potassium channels.