RNA is a biopolymer of high relevance in the biopharmaceuticals field and in fundamental and applied research; however, the preservation of the RNA stability is still a remarkable challenge. Herein, we demonstrate the enhanced potential of aqueous solutions of self-buffering cholinium-based Good's buffers ionic liquids (GB-ILs), at 20 and 50 % (w/w), as alternative preservation media of recombinant small RNAs. The thermal stability of RNA is highly enhanced by GB-ILs, with an increase of 14 °C in the biopolymer melting temperature - the highest increase observed up to date with ILs. Most GB-ILs investigated improve the stability of RNA at least up to 30-days, both at 25 °C and at 4 °C, without requiring the typical samples freezing. Molecular dynamics simulations were applied to better understand the molecular-level mechanisms responsible for the observed RNA improved stability. The number of IL cations surrounding the RNA chain is similar, yet with differences found for the IL anions, which are responsible for the overall charge of the biopolymer first solvation sphere. No cytotoxicity of the studied solutions containing RNA and ILs at 20 % (w/w) was observed onto two distinct human cell lines, reinforcing their potential to act as preservation media when foreseeing biopharmaceutical applications. Finally, RNA was successfully recovered from the ILs aqueous solutions, without changes in its structural integrity, and the ILs successfully recycled and reused.

The co-delivery of multiple chemotherapeutics by micellar delivery systems is a valuable approach to improve cancer treatment since various disease hallmarks can be targeted simultaneously. However, the delivery of multiple drugs requires a nanocarrier structure that can encapsulate various bioactive molecules. In this study, we evaluate the simultaneous encapsulation of a novel triple drug combination in D-α-tocopheryl polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) amphiphilic micelles for cancer therapy. The drug mixture involves two anti-tumoral drugs, Crizotinib and Palbociclib combined with Sildenafil, a compound that is capable of increasing drug accumulation in the intracellular compartment. Such combination aims to achieve an enhanced cytotoxic effect in cancer cells. Our results demonstrated that TPGS-PLA copolymers self-assembled into stable nanosized micelles (158.3 nm) capable of co-encapsulating the three drugs with high loading efficiency. Triple drug loaded TPGS-PLA micelles were internalized in A549 non-small lung cancer cells and exhibited an improved cytotoxic effect in comparison with single (Crizotinib) or dual (Crizotinib–Palbociclib) drug loaded micelles, indicating the therapeutic potential of the triple co-delivery strategy. These findings demonstrate that TPGS-PLA micelles are suitable carriers for multiple drug delivery and also that this particular drug combination may have potential to improve cancer treatment.

Regucalcin (RGN) is a calcium (Ca(2+))-binding protein that displays a characteristic downregulated expression with aging in several tissues. Besides its role in regulating intracellular Ca(2+) homeostasis, RGN has been associated with the control of oxidative stress, cell proliferation, and apoptosis. Thus, the diminished expression of RGN with aging may contribute to the age-associated deterioration of cell function. In the present study, we hypothesized that the maintenance of high expression levels of RGN may prevent age-related alterations in the processes mentioned previously. First, we confirmed that RGN expression is significantly diminished in the prostate of 8-, 9-, 12-, and 24-months wild-type rats. Then, the effect of aging on lipid peroxidation, antioxidant defenses, cell proliferation, and apoptosis in the prostate of wild-type controls and transgenic rats overexpressing RGN (Tg-RGN) was investigated. The activity of glutathione and the antioxidant capacity were increased in Tg-RGN rats in response to the age-associated increase in thiobarbituric acid reactive substances levels, an effect not seen in wild type. Overexpression of RGN also counteracted the effect of aging increasing prostate cell proliferation. In contrast to wild-type animals, the prostate weight of Tg-RGN did not change with aging and was underpinned by the diminished expression of stem cell factor and c-kit, and increased expression of p53. In addition, aged Tg-RGN animals displayed increased expression (activity) of apoptosis regulators, therefore not showing the age-induced resistance to apoptosis observed in wild type. Altogether, these findings indicate the protective role of RGN against the development of age-related pathologies, such as, for example, prostate cancer.

Diabetic nephropathy (DN) is a common complication in patients with diabetes, and most of them need renal replacement therapy such as haemodialysis (HD). These patients have a high tendency to develop infections and exhibit anomalies in the immune system. The objective of this study was to assess the expression of activation-related markers on T cells, as well as to quantify inflammatory cytokines, before and after a single HD session in DN patients. The study involved DN patients under HD treatment who signed an informed consent form. Blood samples before and after one HD session were collected, to analyse the expression of CD25, CD69 and CD71 in T cells. We also quantified IL-12p70, IL-8, IL-10, IL-1β, TNF-α and IL-6 in serum samples using the cytometric bead array technique. After the HD session, there was an increase in the CD4/CD8 ratio due to significant alterations in both subsets. The relative percentage of CD25+ cells and CD8+ CD25+ increased significantly after the HD session, while the relative percentage of CD69 T cells decreased. There was a significant decrease in the CD25 mean fluorescence intensity values for CD4+ T, as well as in the case of CD71 in T cells after the HD session. Regarding cytokine synthesis, we found a significant increase in IL-10 and IL-6 and a decrease in IL-8 after HD session. This study showed that a HD session in DN patients affects the T cell activation status in the two major subpopulations and differentially modulates the production of inflammatory cytokines.

Regucalcin (RGN) is a calcium-binding protein underexpressed in human prostate cancer cases, and it has been associated with the suppression of cell proliferation and the regulation of several metabolic pathways. On the other hand, it is known that the metabolic reprogramming with augmented glycolytic metabolism and enhanced proliferative capability is a characteristic of prostate cancer cells. The present study investigated the influence of RGN on the glycolytic metabolism of rat prostate by comparing transgenic adult animals overexpressing RGN (Tg-RGN) with their wild-type counterparts. Glucose consumption was significantly decreased in the prostate of Tg-RGN animals relatively to wild-type, and accompanied by the diminished expression of glucose transporter 3 and glycolytic enzyme phosphofructokinase. Also, prostates of Tg-RGN animals displayed lower lactate levels, which resulted from the diminished expression/activity of lactate dehydrogenase. The expression of the monocarboxylate transporter 4 responsible for the export of lactate to the extracellular space was also diminished with RGN overexpression. These results showed the effect of RGN in inhibiting the glycolytic metabolism in rat prostate, which was underpinned by a reduced cell proliferation index. The present findings also suggest that the loss of RGN may predispose to a hyper glycolytic profile and fostered proliferation of prostate cells.

Nanocarriers with a pH responsive behavior are receiving an ever growing attention due to their potential for promoting on-demand drug release and thus increase the therapeutic effectiveness of anti-tumoral pharmaceutics. However, the majority of these systems require costly, time-consuming and complex chemical modifications of materials or drugs to synthesize nanoparticles with pH triggered release. Herein, the development of dual drug loaded pH-responsive mesoporous silica nanoparticles (MSNs) with a calcium carbonate-based coating is presented as an effective alternative. This innovative approach allowed the loading of a non-steroidal anti-inflammatory drug (Ibuprofen) and Doxorubicin, with high efficiency. The resulting dual drug loaded MSNs have spherical morphology and a mean size of 171 nm. Our results indicate that under acidic conditions the coating disassembles and the drugs are rapidly released, whereas at physiologic pH the release is slower and gradually increases with time. Furthermore, an improved cytotoxic effect was obtained for Doxorubicin–Ibuprofen MSNs coated with CaCO3 in comparison with non-coated particles. The cytotoxic effect of dual loaded carbonate coated particles, was similar to that of Doxorubicin + Ibuprofen free drug administration at 72 h, even with the delivery of a significantly lower amount of drug by MSNs-CaCO3. These results suggest that the carbonate coating of MSNs is a promising approach to create a pH-sensitive template for a delivery system with application in cancer therapy.

Frequently, skin is subjected to damaging events, such as deep cuts, burns or ulcers, which may compromise the integrity of this organ. To overcome such lesions, different strategies have been employed. Among them, wound dressings aimed to re-establish skin native properties and decreased patient pain have been pursued for a long time. Herein, an electrospun membrane comprised by deacetylated/arginine modified chitosan (CH-A) was produced to be used as a wound dressing. The obtained results showed that the membrane has a highly hydrophilic and porous three-dimensional nanofibrous network similar to that found in human native extracellular matrix. In vitro data indicate that human fibroblasts adhere and proliferate in contact with membranes, thus corroborating their biocompatibility. This nanofiber-based biomaterial also demonstrated bactericidal activity for two bacterial strains. In vivo application of CH-A nanofibers in full thickness wounds resulted in an improved tissue regeneration and faster wound closure, when compared to non-modified membranes. Such findings support the suitability of using this membrane as a wound dressing in a near future.

The development of prostate cancer (PCa) is intimately associated with the hormonal environment, and the sex steroids estrogens have been implicated in prostate malignancy. However, if some studies identified estrogens as causative agents of PCa, others indicated that these steroids have a protective role counteracting prostate overgrowth. The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF), have been associated with the control of cell proliferation/apoptosis and prostate carcinogenesis, and studies show that estrogens regulate their expression in different tissues, though, in the case of prostate this remains unknown. The present study aims to evaluate the role of 17β-estradiol (E2) in regulating the expression of SCF/c-KIT in human prostate cell lines and rat prostate, and to investigate the consequent effects on prostate cell proliferation and apoptosis. qPCR, Western Blot, and immuno(cito)histochemistry analysis showed that E2-treatment decreased the expression of SCF and c-KIT both in human prostate cells and rat prostate. Furthermore, the diminished expression of SCF/c-KIT was underpinned by the diminished prostate weight and reduced proliferation index. On the other hand, the results of TUNEL labelling, the increased activity of caspase-3, and the augmented expression of caspase-8 and Fas system in the prostate of E2-treated animals indicated augmented apoptosis in response to E2. The obtained results demonstrated that E2 down-regulated the expression of SCF/c-KIT system in prostate cells, which was associated with antiproliferative and proapoptotic effects. Moreover, these findings support the protective role of estrogens in PCa and open new perspectives on the application of estrogen-based therapies.

Catechol-O-methyltransferase (COMT; EC 2.1.1.6) is a magnesium-dependent enzyme that catalyzes the methylation reaction of different catecholic substrates such as catecholamines, xenobiotic catechols and catecholestrogens. Following the initial characterizations of these enzymes, it was described that they are potentially involved in diverse human disorders. Specifically, as its inhibition has proven to be of great interest in neurologic disorders such as Parkinson's disease, developing inhibitor molecules with increased potency and selectivity may improve the outcome of these patients. These molecules are usually accomplished using structure-based drug design studies that rely on the attainment of highly purified protein quantities. Indeed, challenges in the determination of protein structures are mainly associated with their low natural abundance coupled with the difficulty of obtaining crystals amenable to X-Ray diffraction. In particular, as membrane proteins are naturally embedded in the lipid bilayer, the determination of their structure faces additional difficulties. As it is unrealistic to purify all of these targets from their natural sources, structural biology of proteins usually focus on the recombinant heterologous expression of these proteins onto an expression host. In addition, to isolate the target proteins from the other major host contaminants, equally appropriated purification strategies need to be designed and implemented, mostly using chromatographic procedures. Throughout this entire process, is also important that the developed strategy is able to keep the proteins in a stable and functional active form, thus avoiding its misfolding during biosynthesis and aggregation after its recovery and isolation in the downstream processing. Therefore, the main scope of this work is the development of a straightforward approach that allows the biosynthesis, isolation and purification of recombinant human COMT isoforms in a biologically active form for further application in structural studies or to evaluate their role as potential therapeutic proteins. Specifically, although no single host can provide all the desired properties for recombinant protein biosynthesis, Pichia pastoris is able to perform many post-translational modifications and is cultivated at high cell-densities in moderately cheap media. Therefore, in this work, it was selected for expression of COMT enzymes. On the other hand, the high selectivity often provided by affinity chromatography prompted us to employ it as the main isolation and purification step. The determination of COMT enzymatic activity is greatly important in COMT recombinant research, either to assess COMT activity from recombinant lysates or purified fractions, for detergent-solubilized or unsolubilized samples and for both isoforms. Therefore, a faster and more sensitive analytical method based on HPLC coupled with coulometric detection was developed for quantifying metanephrine in these assays. Then, an integrated strategy for recombinant soluble catechol-O-methyltransferase (SCOMT) biosynthesis onto P. pastoris and purification using immobilized-metal affinity chromatography was implemented where highly purified fractions of this target enzyme were obtained. On the other hand, as heterologous membrane protein overexpression is usually more challenging than soluble proteins and less reports are available in the literature with recombinant human membrane-bound catechol-O-methyltransferase (MBCOMT) than COMT soluble isoform, our work were mostly focused on MBCOMT. Here, we established protocols for MBCOMT expression in Pichia pastoris methanol-induced cultures in baffled shake-flasks and mini-bioreactors. In particular, the optimization of the induction phase using artificial neural networks in mini-biorreactors allowed achieving high levels of biologically active MBCOMT. Then, arginine-affinity chromatography was successfuly applied for the direct capture of MBCOMT from Pichia pastoris lysates and it was recovered in a moderate purified form. Finally, the ongoing work is related to the purification of a hexa-histidine tagged form of MBCOMT using immobilized-metal affinity chromatography. Indeed, despite significant achievements were made concerning the construction of a tagged form of MBCOMT solubilized with an appropriated detergent in a biologically active form, additional stepwise gradients are required to effectively separate MBCOMT from the other contaminants. In conclusion, the progress achieved with this work meets the highly demanding requirements of biophysical techniques, mainly regarding the upstream stage as well as COMT stabilization where moderate to high quantities of catalitically active enzymes were obtained. In particular, coupling the strategy here reported for SCOMT with a final polishing step will probably allow performing structural or bio-interaction studies with this enzyme. Nonetheless, the strategies here described successfully for partial MBCOMT purification need to be improved, especially for immobilized-metal affinity chromatography once it is considered to be highly selective and, thus, it is feasible that after succesful optimization procedures, fractions with high purity will be obtained. Therefore, the strategies here reported with the intensification and optimization of some procedures would possible permit performing structural and bio-interaction studies using the apo-enzymes or complexed with different ligands (cofactors or inhibitors) by Nuclear Magnetic Ressonance, Isothermal Titration Calorimetry or even using Crystallographic experiments.