Background/Objectives: Flavonoids are low-molecular-weight polyphenolic compounds that are universally distributed in plants. They are a chemically varied group of secondary metabolites with a broad range of biological activity. The use of flavonoids is known to decrease the risk of many chronic diseases due to their radical scavenging, antioxidant, anti-inflammatory, anticarcinogenic, and antimutagenic properties. Limitations in the use of flavonoids include their low water solubility and poor stability, and therefore their low bioavailability. The encapsulation of flavonoids in different nanocarriers has helped to overcome this limitation. Taking this into account, in this work, the encapsulation of four flavones with several therapeutic applications—7-hydroxyflavone, 7,8-dihydroxyflavone, baicalein, and luteolin—in poly(lactic-co-glycolic) acid (PLGA)-derived polymeric nanoparticles (NPs) has been investigated. Methods: A physicochemical characterization of the NPs has been carried out using different techniques, including the evaluation of antioxidant and antimicrobial activities. Results: In all cases, the encapsulation efficiency of the four flavones in the prepared NPs was high (>90%), the zeta potential was about −31 mV, and the size was nanometric (~450 nm). The drug release from the nanoparticles was also studied, showing first-order kinetics. Statistical tools were applied to the release rate constants. The antioxidant activity and the in vitro antimicrobial activity of the free and flavone-loaded NPs were investigated, in the case of the latter using Gram-positive and Gram-negative bacteria. Results show that when the flavones are encapsulated, they retain their therapeutic properties. Conclusions: In summary, PLGA-based NPs not only prevent flavone degradation but also significantly boost solubility, ultimately optimizing bioavailability. Our results underscore these NPs as a promising platform for efficient flavone delivery.

Nanoparticles (NPs) have revolutionized the field of medicine and veterinary for both detection and treatment of infection diseases, however, their utilization to supply drugs or bioactive metabolites to marine invertebrates has been insufficiently investigated. Antimicrobial peptides (AMPs) have gained increasing attention as potential alternatives to overcome the shortcomings associated with conventional therapeutic strategies. Using an emulsion/evaporation approach, spherical poly(lactic-co-glycolic acid) (PLGA) NPs charged with D-Caerin (D-CAE) peptide have been prepared. The obtained NPs showed a bimodal size distribution, with mean diameters of 130 +/- 4 and 421 +/- 19 nm. Among the tested formulation, NPs with a peptide/polymer ratio of 0.02 exhibited the best physico-chemical properties, achieving encapsulation efficiencies of 70 %. Release studies demonstrated that 73.5 % of the peptide was liberated from loaded the NPs within 40 h. Furthermore, the D-CAE peptide retained its antimicrobial activity following encapsulation in PLGA NPs and exhibited bactericidal effects against two prevalent pathogens in aquaculture species. Finally, it has been shown that peptide-loaded NPs are effectively ingested by clam post-larvae, suggesting the potential of peptide-PLGA NPs as biocompatible nanocarriers for delivery of AMPs into bivalves and other invertebrates.

Nanotubes are one-dimensional tubular structures characterized by nanoscale radial dimensions and micrometer-scale axial lengths. Their hollow inner cavity confers a high specific surface area and distinctive physicochemical properties, enabling remarkable potential in drug delivery, disease diagnostics, and tissue engineering. Extensive research into their biocompatibility and degradability establishes a solid foundation for further development and rational design. A synthesis of current data across relevant systems, including carbon nanotubes (CNTs), boron nitride nanotubes (BNNTs), titanium dioxide nanotubes (TiNTs), halloysite nanotubes (HNTs), anodic aluminium oxide nanotubes (AAOs), lipid nanotubes (LNTs), and polymeric nanotubes (PNTs), reveals how structural features and physicochemical properties govern biological responses and functional performance. This integration supports a unified "structure-property-bioresponse-function" framework, offering a conceptual roadmap and general design principles to guide the next generation of nanotube-based biomaterials.

The overuse of antibiotics over decades has led to a multi-resistance of bacteria that today poses a serious threat to human health. (3-lactam antibiotics are one of the most prescribed drugs against bacterial infectious diseases, which has conducted to increased resistance. The short half-life of these drugs results in low bioavailability, which limits their clinical use and requires continuous administration by infusion. Encapsulation of these antibiotics in nanocarriers would improve their biopharmaceutical properties by protecting them. With this in mind, single- and multi-walled carbon nanotubes (SWCNTs and MWCNTs, respectively) were used here as nanocarriers for the antibiotics Meropenem and Cefepime. The adsorption process of these antibiotics in the carbon nanotubes (CNTs) was optimized to obtain a high encapsulation efficiency. The complexes CNT/drug prepared were characterized by dynamic light scattering and spectroscopic measurements. The growth of Escherichia coli and Staphylococcus aureus bacteria was analyzed in the presence of the CNT/drug complexes to evaluate the pharmacological properties of the encapsulated antibiotics. Results showed lower minimum inhibitory concentration (MIC) values of the CNT/drug complexes compared to free drugs. This indicates the preservation of the pharmacological properties of the encapsulated antibiotics. In addition, the stability of the encapsulated antibiotics was observed to last at least 24 h, which was a great improvement compared to the free drugs.

Flavonoids are secondary plant metabolites with a wide range of pharmacological effects. Among others, their antioxidant, anti-inflammatory and vasoprotective actions are noteworthy. However, the low bioavailability of flavonoids limits their direct clinical use. Nanoencapsulation of flavonoids is an effective tool to improve their biopharmaceutical characteristics, as the drug is protected inside the nanocarrier and specifically released into the therapeutic target. Bearing this in mind, pristine single- and multi-walled carbon nanotubes have been studied in this work as nanocarriers of 7,8-dihydroxyflavone, 7,8-DHF. Flavone encapsulation is optimized according to the influence of pH, the type of CNTs and their concentration on the association process. The equilibrium binding constants of 7,8-DHF to the CNTs are determined by measuring the variation observed in the flavone absorbance when the concentration of carbon nanotubes increases. CNTs/flavone complexes are characterized by particle size distribution and Z-potential measurements, as well as Transmission Electron Microscopy. The antioxidant capacity of free 7,8-DHF and the CNTs/7,8-DHF complexes are estimated by using the DPPH & sdot;, 2,2-diphenyl-1-picrylhydrazyl radical scavenging method. The results show that the encapsulation of the flavone in the CNTs results in a preservation of its pharmacological properties and provides stability to the encapsulated drug.

The HIV attacks the immune system provoking an infection that is considered a global health challenge. Despite antiretroviral treatments being effective in reducing the plasma viral load in the blood to undetectable levels in people living with HIV (PLWH), the disease is not cured and has become chronic. This happens because of the existence of anatomical and cellular viral reservoirs, mainly located in the lymph nodes and gastrointestinal tract, which are composed of infected CD4+ T cells with a resting memory phenotype and inaccessible to antiretroviral therapy. Herein, a new therapeutic strategy based on nanotechnology is presented. Different combinations of antiretroviral drugs (bictegravir/tenofovir/emtricitabine and nevirapine/tenofovir/emtricitabine) and toll-like receptor agonists were encapsulated into metal-organic frameworks (MOFs) PCN-224 and ZIF-8. The encapsulation efficiencies of all the drugs, as well as their release rate from the carriers, were measured. In vitro studies about the cell viability, the hemocompatibility, and the platelet aggregation of the MOFs were carried out. Epifluorescence microscopy assays confirmed the ability of ZIF-8 to target a carboxyfluorescein probe inside HeLa cell lines and PBMCs. These results pave the way for the use of these structures to eliminate latent HIV reservoirs from anatomical compartments through the activation of innate immune cells, and a higher efficacy of the triplet combinations of antiretroviral drugs.

The goal of this work is to investigate if the synergistic antifungal activity between cyclosporine A, CsA, and voriconazole, VRZ, increases when both drugs are encapsulated in a nanocarrier as compared when they are free. The preparation and characterization of blank and VRZ and CsA loaded polymeric based PLGA nanoparticles (PLGA, PLGA-PEG, and PLGA+PEG) was a necessary previous step. Using the more suitable NPs, those of PLGA, the antifungal susceptibility tests performed with VRZ-loaded PLGA NPs, show no significant increase of the antifungal activity in comparison to that of free VRZ. However, the synergistic behavior found for the (VRZ+CsA)-loaded PLGA NPs was fourfold stronger than that observed for the two free drugs together. On the other hand, the investigation into the suppression of C. albicans biofilm formation showed that blank PLGA NPs inhibit the biofilm formation at high NPs concentrations. However, a minor effect or even a slight biofilm increase formation was observed at low and moderate NPs concentrations. Therefore, the enhancement of the biofilm inhibition found for the three tested treatments (CsA alone, VRZ alone, and VRZ+CsA) when comparing free and encapsulated drugs, within the therapeutic window, can be attributed to the drug encapsulation approach. Indeed, polymeric PLGA NPs loaded with CsA, VRZ, or VRZ+CsA are more effective at inhibiting the C. albicans biofilm growth than their free counterparts.

Vibriosis is one of the most usual infection diseases in bivalve mollusks, particularly affecting seeds and larvae, which are more susceptible than adults to these bacterial infections. The devastating effect of vibriosis in shellfish hatcheries is aggravated by the increasing resistance of many Vibrio species to traditional antibiotics, highlighting the need to find new antimicrobial agents. Antimicrobial peptides (AMPs) are a promising alternative, however, their low stability can be a handicap for their practical application. In this study, the 3D structure and the stability of a synthetic all-D-amino acid peptide (D-Caerin) to proteases and extreme pH conditions have been analyzed and compared with its corresponding natural L-enantiomer peptide, Caerin 1.1. Moreover, the antimicrobial activity of D-Caerin has been tested in vitro against the control bacteria Micrococcus luteus CECT 245; and four Vibrio species: Vibrio aestuarianus CECT 625 T, Vibrio anguillarum CECT 522 T, Vibrio harveyi CECT 525 T and Vibrio tapetis CECT 4600 T, which are among the most representative causative agents of vibriosis in aquaculture. Our results demonstrate that D-Caerin contains two left-handed alpha helices, and is more stable and effective against the bacterial species tested than its corresponding natural L- counterpart. The bioactivity of D-Caerin has also been tested in vivo, in clam seeds infected with a mixed inoculum of these vibrio species. These preliminary assays show that D-Caerin-treated specimens exhibit higher survival rates after infection, demonstrating the superior stability of D-Caerin and its effectiveness against vibriosis-causative agents.

The design and preparation of new vectors to transport genetic material and increase the transfection efficiency continue being an important research line. Here, a novel biocompatible sugar-based polymer derived from D-mannitol has been synthesized to be used as a gene material nanocarrier in human (gene transfection) and microalga cells (transformation process). Its low toxicity allows its use in processes with both medical and industrial applications. A multidisciplinary study about the formation of polymer/p-DNA polyplexes has been carried out using techniques such as gel electrophoresis, zeta potential, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy. The nucleic acids used were the eukaryotic expression plasmid pEGFP-C1 and the microalgal expression plasmid Phyco69, which showed different behaviors. The importance of DNA supercoiling in both transfection and transformation processes was demonstrated. Better results were obtained in microalga cells nuclear transformation than in human cells gene transfection. This was related to the plasmid's conformational changes, in particular to their superhelical structure. It is noteworthy that the same nanocarrier has been used with eukaryotic cells from both human and microalga.

Gene therapy is a technique that is currently under expansion and development. Recent advances in genetic medicine have paved the way for a broader range of therapies and laid the groundwork for next-generation technologies. A terminally substituted difluorene-diester Schiff Base calix[4]arene has been studied in this work as possible nanovector to be used in gene therapy. Changes to luminescent behavior of the calixarene macrocycle are reported in the presence of ct-DNA. The calixarene macrocycle interacts with calf thymus DNA (ct-DNA), generating changes in its conformation. Partial double-strand denaturation is induced at low concentrations of the calixarene, resulting in compaction of the ct-DNA. However, interaction between calixarene molecules themselves takes place at high calixarene concentrations, favoring the decompaction of the polynucleotide. Based on cytotoxicity studies, the calixarene macrocycle investigated has the potential to be used as a nanovehicle and improve the therapeutic efficacy of pharmacological agents against tumors.

Gene therapy is a technique to treat or prevent diseases through the use of therapeutic genetic material. Viral or non-viral vectors are needed to deliver genes into cells. Even if viral vectors show several advantages (long-term gene expression and/or high transfection efficiency), their use could produce side effects in the patients. Therefore, non-viral vectors (with a large-scale production and a low immunogenicity) have been shown to be a good choice as nanovehicles of DNA or RNA. Cationic surfactants form organized structures (liposomes, micelles vesicles, or nanoparticles) that can act as non-viral vectors. Both electrostatic and hydrophobic interactions with the nucleic acids produce a conformational change in their doubled helical structure. This new cationic complex (surfactant-based nanostructures + DNA) crosses the lipid bilayer of the cells and delivers the genetic material into the cytosol. In the last years, nanostructures containing cationic metallosurfactants have been successfully used as non-viral vectors. These surfactants contain metal ions incorporated into their structure, generally in their hydrophilic head groups. Redox properties of these metals could be utilized to optimize the transfection process through their self-assembly. The hydrophobic nature of the surfactants also influences the gene delivery. Metallosurfactants can also participate in biological processes such as metabolisms involving enzymes or the production of radical species with antimicrobial properties.

The research on flavonoids has exponentially grown since their first therapeutic evidence, in 1937. They are effective in vitro in a wide range of human diseases, particularly those mediated by free radicals, such as cancer, atherosclerosis, AIDS, or neuronal diseases. However, their applications have been reduced due to their low solubility, poor absorption, and rapid metabolism. Flavonoid encapsulation in nanocarriers significantly improves their oral absorption, protects the drug against degradation, decreases the first-pass hepatic effect, and makes absorption through the lymphatic system easier. In this work, carbon nanotubes were used as nanocarriers of 7-hydroxyflavone, 7-HF. The encapsulation of 7-HF into pristine single- and multi-walled carbon nanotubes, and into -COOH functionalized single-walled carbon nanotubes has been investigated. The equilibrium association constants were estimated. The structural backbone of 7-HF, two benzene rings linked through three carbon atoms that form a pyran heterocyclic ring containing a keto group, seems to play a key role in the 7-HF/CNT interactions, although other types of interactions are also at work. The in vitro release of 7-HF was studied at three pHs, 2.0, 7.4, and 9.2, mimicking the different biological barriers of the human organism.

Polyplexes formed between a cationic polymer, PUArab, and both linear and plasmid DNA were studied. The transfection efficiency of PURarab/pDNA was investigated.

The obtaining of nanocarriers of gene material and small drugs is still an interesting research line. Side-effects produced by the toxicity of several pharmaceutics, the high concentrations needed to get therapeutic effects, or their excessive use by patients have motivated the search for new nanostructures. For these reasons, cationic metallo-liposomes composed by phosphatidylcholine (PC), cholesterol (CHO) and RuC1C19 (a surfactant derived from the metallic complex [Ru(bpy)3]2+) were prepared and characterized by using diverse techniques (zeta potential, dynamic light scattering and electronic transmission microscopy –TEM-). Unimodal or bimodal populations of spherical aggregates with small sizes were obtained depending on the composition of the liposomes. The presence of cholesterol favored the formation of small aggregates. ct-DNA was condensed in the presence of the liposomes investigated. In-vitro assays demonstrated the ability of these nanoaggregates to internalize into different cell lines. A positive gene transfection into human bone osteosarcoma epithelial cells (U2OS) was also observed. The RuC1C19 surfactant was used as sensor to quantify the binding of DNA to the liposomes. Doxorubicin was encapsulated into the metallo-liposomes, demonstrating their ability to be also used as nanocarriers of drugs. A relationship between then encapsulation percentage of the antibiotic and the composition of the aggregates has been established.

The interaction (adsorption process) of commercial ionic surfactants with non-functionalized and functionalized carbon nanotubes (CNTs) has been studied by potentiometric measurements based on the use of ion-selective electrodes. The goal of this work was to investigate the role of the CNTs’ charge and structure in the CNT/surfactant interactions. Non-functionalized single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), and amine functionalized SWCNT were used. The influence of the surfactant architecture on the CNT/surfactant interactions was also studied. Surfactants with different charge and hydrophobic tail length (sodium dodecyl sulfate (SDS), octyltrimethyl ammonium bromide (OTAB), dodecyltrimethyl ammonium bromide (DoTAB) and hexadecyltrimethyl ammonium bromide (CTAB)) were studied. According to the results, the adsorption process shows a cooperative character, with the hydrophobic interaction contribution playing a key role. This is made evident by the correlation between the free surfactant concentration (at a fixed [CNT]) and the critical micellar concentration, cmc, found for all the CNTs and surfactants investigated. The electrostatic interactions mainly determine the CNT dispersion, although hydrophobic interactions also contribute to this process.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

The interaction between calf-thymus DNA, ctDNA, and various single-chained surfactants with different functional groups at the end of hydrophobic tail was studied with the goal of investigating the influence of the functional group nature on surfactant DNA compacting efficiency. The surfactants investigated were dodecyltriethylammonium bromide (DTEABr), triethyl(1-phenoxydodecyl)ammonium bromide (12PhBr), triethyl(2-naphthoxydodecyl)ammonium bromide (12NBr) and 11-(isonicotinoyloxy)-N,N,N-triethyl-1-undecanaminium bromide (11PyBr). Results made evident that the surfactants’ tendencies to self-aggregation is the key factor determining their efficiency to compact the nucleic acid. Subsequently, DOPE/12NBr/pEGFP-C1 lipoplexes, with different cationic surfactant molar fractions (α) and mass ratios (L/D), were prepared and characterized. DOPE is a zwitterionic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the plasmid pEGFP-C1 carries a GFP coding sequence with the necessary regulatory elements for constitutive expression of the gene in human cells. 12NBr was chosen because it was the most efficient DNA compacting agent among the surfactants investigated. Finally, the cytotoxicity and transfection efficiency (TE) of DOPE/12NBr/pDNA lipoplexes, with different compositions, were investigated.

Aquaculture is threatened by severe bacterial and viral outbreaks which can cause massive mortality and limit productivity. Antimicrobial peptides are a promising tool for the control of infectious diseases; however, their use in the aquaculture sector has been poorly explored. Recombinant production of peptides is an interesting alternative to their chemical synthesis, which has still important challenges to address. Although extra amino acid tags are usually added to the ends of recombinant proteins to enhance their stability and facilitate their purification or their translational processing, the effect of these tags on the function and the stability of antimicrobial peptides is completely unknown. This is particularly important in the case of the short cationic antimicrobial peptide caerin, originally isolated from frog skin secretions, and other peptides which bioactivity has been directly related with its ability to adopt an amphipathic alpha-helical structure upon interaction with the microbial membranes. Caerin has demonstrated bactericidal activity against several important human pathogens, however its antimicrobial activity over usual aquaculture pathogens remains practically unexplored. In the present work, we have studied the effect of adding the terminal tag polyhistidine (6xHis) and the 2A peptide from the foot and mouth disease virus (FMDV-2A) to the N- or the C- terminus of caerin on its activity against a collection of bacterial and viral pathogens common to aquaculture species. In addition, the antimicrobial activity of a peptide composed of two tandem consecutive caerin sequences has been investigated. We have concluded that the addition of terminal tags to caerin causes a drastic reduction of its antiviral activity against the studied viruses and that the effect of the added tags on the bactericidal activity of the peptide depends of the target bacterial species. However, caerin with a 6xHis extension at its N terminus maintains or increases its antimicrobial activity against most of the fish pathogenic bacteria tested. According to our data, there is only a moderate agreement between the theoretical predictions obtained from in silico models and the experimental observations. This information is essential for the rational design of strategies to aim the recombinant production of caerin and other antimicrobial peptides, which can be used as anti-infective agents in aquaculture.

Gene therapy is a therapeutic process consisting of the transport of genetic material into cells. The design and preparation of novel carriers to transport DNA is an important research line in the medical field. Hybrid compounds such as metallo-liposomes, containing a mixture of lipids, were prepared and characterized. Cationic metal lipids derived from the [Ru(bpy)3]2+ complex, RuC11C11 or RuC19C19, both with different hydrophobic/lipophilic ratios, were mixed with the phospholipid DOPE. A relation between the size and the molar fraction α was found and a multidisciplinary study about the interaction between the metallo-liposomes and DNA was performed. The metallo-liposomes/DNA association was quantified and a relationship between Kapp and α was obtained. Techniques such as AFM, SEM, zeta potential, dynamic light scattering and agarose gel electrophoresis demonstrated the formation of lipoplexes and showed the structure of the liposomes. L/D values corresponding to the polynucleotide’s condensation were estimated. In vitro assays proved the low cell toxicity of the metallo-liposomes, lower for normal cells than for cancer cell lines, and a good internalization into cells. The latter as well as the transfection measurements carried out with plasmid DNA pEGFP-C1 have demonstrated a good availability of the Ru(II)-based liposomes for being used as non-toxic nanovectors in gene therapy.