Studies of Protein Binding to Biomimetic Membranes Using a Group of Uniform Materials Based on Organic Salts Derived From 8-Anilino-1-naphthalenesulfonic Acid.

Tuning the 8-anilino-1-naphthalenesulfonic acid (ANS) structure usually requires harsh conditions and long reaction times, which can result in low yields. Herein, ANS was modified to form an ANS group of uniform materials based on organic salts (GUMBOS), prepared with simple metathesis reactions and distinct cations, namely tetrabutylammonium (N4444), tetrahexylammonium (N6666), and tetrabutylphosphonium (P4444). These ANS-based GUMBOS were investigated as fluorescent probes for membrane binding studies with four proteins having distinct physicochemical properties. Liposomes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine were employed as membrane models as a result of their ability to mimic the structure and chemical composition of cell membranes. Changes in fluorescence intensity were used to monitor protein binding to liposomes, and adsorption data were fitted to a Freundlich-like isotherm. It was determined that [N4444][ANS] and [P4444][ANS] GUMBOS have enhanced optical properties and lipophilicity as compared to parent ANS. As a result, these two GUMBOS were selected for subsequent protein-membrane binding studies. Both [N4444][ANS] and [P4444][ANS] GUMBOS and parent ANS independently reached membrane saturation within the same concentration range. Furthermore, distinct fluorescence responses were observed upon the addition of proteins to each probe, which demonstrates the impact of properties such as lipophilicity on the binding process. The relative maintenance of binding cooperativity and maximum fluorescence intensity suggests that proteins compete with ANS-based probes for the same membrane binding sites. Finally, this GUMBOS-based approach is simple, rapid, and involves relatively small amounts of reagents, making it attractive for high-throughput purposes. These results presented herein can also provide relevant information for designing GUMBOS with ameliorated properties.

Vanadium Compounds with Antidiabetic Potential.

Over the last four decades, vanadium compounds have been extensively studied as potential antidiabetic drugs. With the present review, we aim at presenting a general overview of the most promising compounds and the main results obtained with in vivo studies, reported from 1899-2023. The chemistry of vanadium is explored, discussing the importance of the structure and biochemistry of vanadate and the impact of its similarity with phosphate on the antidiabetic effect. The spectroscopic characterization of vanadium compounds is discussed, particularly magnetic resonance methodologies, emphasizing its relevance for understanding species activity, speciation, and interaction with biological membranes. Finally, the most relevant studies regarding the use of vanadium compounds to treat diabetes are summarized, considering both animal models and human clinical trials. An overview of the main hypotheses explaining the biological activity of these compounds is presented, particularly the most accepted pathway involving vanadium interaction with phosphatase and kinase enzymes involved in the insulin signaling cascade. From our point of view, the major discoveries regarding the pharmacological action of this family of compounds are not yet fully understood. Thus, we still believe that vanadium presents the potential to help in metabolic control and the clinical management of diabetes, either as an insulin-like drug or as an insulin adjuvant. We look forward to the next forty years of research in this field, aiming to discover a vanadium compound with the desired therapeutic properties.

Realism-based assessment of the efficacy of potassium peroxymonosulphate on Stenotrophomonas maltophilia biofilm control.

The potential of pentapotassium bis(peroxymonosulphate) bis(sulphate) (OXONE) to control biofilms in drinking water distribution systems (DWDS) was evaluated and compared to chlorine disinfection. Mature biofilms of drinking water (DW)-isolated Stenotrophomonas maltophilia were formed using a simulated DWDS with a rotating cylinder reactor (RCR). After 30 min of exposure, OXONE at 10 × minimum bactericidal concentration (MBC) caused a significant 4 log reduction of biofilm culturability in comparison to the unexposed biofilms and a decrease in the number of non-damaged cells below the detection limit (4.8 log cells/cm2). The effects of free chlorine were restricted to approximately 1 log reduction in both biofilm culturability and non-damaged cells. OXONE in synthetic tap water (STW) at 25 ºC was more stable over 40 days than free chlorine in the same conditions. OXONE solution exhibited a disinfectant decrease of about 10% of the initial concentration during the first 9 days, and after this time the values remained stable. Whereas possible reaction of chlorine with inorganic and organic substances in STW contributed to free chlorine depletion of approximately 48% of the initial concentration. Electron paramagnetic resonance (EPR) spectroscopy studies confirmed the presence of singlet oxygen and other free radicals during S. maltophilia disinfection with OXONE. Overall, OXONE constitutes a relevant alternative to conventional DW disinfection for effective biofilm control in DWDS.

A New Microwave-Assisted Protocol for Cellulose Extraction from Eucalyptus and Pine Tree Wood Waste.

An enormous interest in the development of efficient protocols for cellulose extraction has been demonstrated in the last few years, although usually based on non-sustainable chemical and thermal approaches. In this work, we propose a new and more sustainable method for cellulose extraction from eucalyptus and pine tree wood waste products exclusively performed using microwave-assisted radiation. The methodology includes three main steps: (i) alkaline treatment; (ii) bleaching I, using H2O2; and (iii) bleaching II, an acidic treatment. Samples obtained in each step were characterized by Fourier-transform Infrared (FTIR) spectroscopy, powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results were compared with the structural and thermal profile of the starting materials, a commercially available microcrystalline cellulose and with an industrial paper pulp sample. Results confirmed that for both types of wood wastes, cellulose was retained during the extraction procedures and that the removal of hemicellulose and lignin was mainly achieved in the last step, as seen by the FTIR spectra and TGA curves. The developed protocol is innovative, as it constitutes an easy and quick approach for extracting cellulose from eucalyptus and pine tree wood waste. Mild chemical and thermal conditions are used during the three extraction steps (microwave irradiation, aqueous solutions, maximum of 120 °C in a total of 3 h). Moreover, environmentally friendly purification steps are applied based on the use of water and ethanol. This approach offers the possibility of a future scale-up study to potentially apply the developed protocol to the extraction of cellulose on an industrial scale.

A combined experimental and computational study to discover novel tyrosinase inhibitors.

Depigmenting properties of tyrosinase inhibitors (TAi) boosted the search for new compounds applicable in cosmetics. Kojic acid, a 3-hydroxy-4-pyrone, is the most studied tyrosinase inhibitor but undesirable side effects, like dermatitis, and unspecified mechanism led to its exclusion in several countries. To discover safer and more efficient TA, we evaluated tyrosinase inhibitory effect of twelve 3-hydroxy-4-pyridinones (3,4-HPO) in vitro and considering the two reaction steps of inhibition in mushroom tyrosinase enzyme. In parallel we performed molecular docking studies in human and mushroom enzymes. Ligands I6 and I11 were the most effective compounds considering their inhibitory activity in both reaction steps. Our studies revealed that I6 has a non-competitive and mixed type of inhibition for monophenolase and diphenolase activity, while ligand I11 showed a mixed and competitive inhibition type for each reaction step. Molecular Docking results indicated that ligands tend to bind the enzyme by coordinating directly with the binuclear cooper centre and highlighted the relevance of voluminous and non-polar substituents at R2 to avoid the binding of the ligands to the enzyme. The work clarifies the type of inhibition established for kojic acid and points out the differences found for the set of 3,4-HPO chelators studied as prospective tyrosinase inhibitors.

Polymeric Microneedles for Transdermal Delivery of Rivastigmine: Design and Application in Skin Mimetic Model.

In the last years, microneedles (MNs) have been considered a valuable, painless, and minimally invasive approach for controlled transdermal drug delivery (TDD). Rivastigmine (RV), a drug administered to patients suffering from dementia, is currently delivered by oral or transdermal routes; however, both present limitations, mainly gastrointestinal adverse symptoms or local skin irritation and drug losses, respectively, for each route. Given this, the objective of the present work was to develop and evaluate the potential of polymeric MNs for RV transdermal delivery in a controlled manner. Polymeric MNs with two needle heights and different compositions were developed with calcein as a fluorescent model molecule. Morphology and mechanical characterisation were accessed. Skin permeation experiments showed the ability of the devices to deliver calcein and confirmed that the arrays were able to efficiently pierce the skin. To obtain a new TDD anti-dementia therapeutic solution, RV was loaded in 800 µm polymeric MNs of alginate and alginate/k-carrageenan MNs. In the presence of RV, the MN's morphology was maintained; however, the presence of RV influenced the compression force. Skin permeation studies revealed that RV-loaded MNs allowed a more efficient controlled release of the drug than the commercial patch. In vivo, skin irritation tests in rabbits revealed that the developed MNs were innocuous upon removal, in contrast with the evidence found for Exelon®, the commercial patch, which caused slight mechanical damage to the skin. The herein-produced MNs demonstrated a more controlled release of the drug, being the more suitable option for the transdermal delivery of RV.

Sequential injection method for bi-parametric determination of iron and manganese in soil leachates.

The aim of this work was to develop a sequential injection (SI) method for the determination of the micronutrients iron and manganese, in soil leachates, as a tool to assess potential groundwater contamination. The described sequential injection method was based on the reaction of iron with chelator MRB12, a greener alternative chromogenic reagent, and the reaction of manganese with zincon, within a single manifold. The developed SI method enabled the determination of iron in the range 0.10-1.00 mg L-1, and manganese in the range 0.25-2.5 mg L-1 with a limit of detection of 0.08 mg L-1 for iron and 0.24 mg L-1 for manganese. The determination of both parameters was made in 6 minutes, in triplicate. The application to monitor laboratory scale soil core columns (LSSCs), as a simulation of the soil leaching process, proved its efficiency to assess potential contamination of ground waters. Iron and manganese contents were effectively analysed in two different scenarios to mimic the leaching process with rainwater and fertilizer.

Protocol for the Isolation of Stratum Corneum from Pig Ear Skin: Evaluation of the Trypsin Digestion Conditions.

Stratum corneum (SC) represents the outermost layer of the skin, being an effective barrier against the entry of molecules and pathogens. Skin research has given particular focus to SC as it hampers effective drug delivery for cosmetical and therapeutical purposes. Following recommendations to develop alternative models to animals, the SC isolated from skin obtained from medical procedures or from pigs has gained extensive attention. Yet, there is still missing a standard and simple procedure accepted within the scientific community to avoid application of different isolated SC methodologies, a fact that may hamper progress in skin research. Considering this challenge, the present study evaluated different experimental conditions aiming to establish a useful and sustainable solvent-free procedure for the obtention of a realistic SC model. The studied trypsin digestion parameters included concentration, incubation period and temperature. Isolated SC was characterized using histological analysis and calcein's permeability, after the procedure and during a 6-week storage period. Data recommend trypsin digestion at 4 °C for 20 h as the most effective procedure to isolate SC from pig ear skin. This work contributes to standardize the SC isolation procedure, and to obtain a valuable and reliable SC mimetic model for skin drug development.

Marine polymeric microneedles for transdermal drug delivery.

Transdermal drug delivery is considered one of the most attractive routes for administration of pharmaceutic and cosmetic active ingredients due to the numerous advantages, especially over oral and intravenous methodologies. However, some limitations still exist mainly regarding the need to improve the drugs permeation across the skin. For this, several strategies have been described, considering the application of chemical permeation enhancers, drugs' nanoformulations and physical methods. Of these, microneedles have been proposed in the last years as promising strategies to enhance transdermal drug delivery. In this review, different types of microneedles are described, and the most commonly used methods of fabrication systematized, as well as the materials typically used and their main therapeutical applications. A special attention is paid to polymeric microneedles, particularly those made from sustainable marine polysaccharides like chitosan, alginate and hyaluronic acid. The applications of marine based polymeric microneedle devices for transdermal drug delivery are examined in detail and the perspectives of translation from the clinical trials to the market demonstrated.

Foliar application of 3-hydroxy-4-pyridinone Fe-chelate [Fe(mpp)3 ] induces responses at the root level amending iron deficiency chlorosis in soybean.

Iron (Fe) deficiency chlorosis (IDC) affects the growth of several crops, especially when growing in alkaline soils. The application of synthetic Fe-chelates is one of the most commonly used strategies in IDC amendment, despite their associated negative environmental impacts. In a previous work, the Fe-chelate tris(3-hydroxy-1-(H)-2-methyl-4-pyridinonate) iron(III) [Fe(mpp)3 ] has shown great potential for alleviating IDC in soybean (Glycine max) in the early stages of plant development under hydroponic conditions. Herein, its efficacy was verified under soil conditions in soybean grown from seed to full maturity. Chlorophyll levels, plant growth, root and shoot mineral accumulation (K, Mg, Ca, Na, P, Mn, Zn, Ni, and Co) and FERRITIN expression were accessed at V5 phenological stage. Compared to a commonly used Fe chelate, FeEDDHA, supplementation with [Fe(mpp)3 ] led to a 29% higher relative chlorophyll content, 32% higher root biomass, 36% higher trifoliate Fe concentration, and a twofold increase in leaf FERRITIN gene expression. [Fe(mpp)3 ] supplementation also resulted in increased accumulation of P, K, Zn, and Co. At full maturity, the remaining plants were harvested and [Fe(mpp)3 ] application led to a 32% seed yield increase when compared to FeEDDHA. This is the first report on the use of [Fe(mpp)3 ] under alkaline soil conditions for IDC correction, and we show that its foliar application has a longer-lasting effect than FeEDDHA, induces efficient root responses, and promotes the uptake of other nutrients.

Design and Characterization of Sodium Alginate and Poly(vinyl) Alcohol Hydrogels for Enhanced Skin Delivery of Quercetin.

Nature has led to the discovery of biopolymers with noteworthy pharmaceutical applications. Blended biopolymers have demonstrated promising characteristics when compared with their individual counterparts. Sodium alginate (SA) is a marine polymer that has demonstrated the ability to form hydrogels, an interesting property for the development of cutaneous formulations. Predicting the good performance of blended biopolymers, a novel series of hybrid hydrogels based on SA and poly(vinyl) alcohol (PVA) were prepared. Quercetin, a natural polyphenolic flavonoid commonly found in fruits and vegetables, is widely known for its strong anti-inflammatory and antioxidant activity, thus with potential applications against melanoma, dermatitis, psoriasis, and skin ageing. Here, hydrogels were produced at different ratios of SA and PVA. The surface morphology, structure, interaction of polymers, the capacity to absorb water and the entrapment efficiency of quercetin were evaluated for the blended hydrogels. Targeting the cutaneous application of the formulations, the rheological properties of all unloaded and quercetin-loaded hydrogels revealed pseudoplastic behavior, evidence of non-thixotropy, good resistance to deformation, and profile maintenance with temperatures ranging from 20 °C up to 40 °C. The incorporation of quercetin in the hydrogel retained its antioxidant activity, confirmed by radical scavenging assays (ABTS and DPPH). The permeability of quercetin through the skin showed different penetration/permeation profiles according to the hydrogel's blend. This behavior will allow the selection of SA-PVA at 2/1 ratio for a local and prolonged skin effect, making the use of these hydrogels a good solution to consider for the treatment of skin ageing and inflammation.

Application of the human stratum corneum lipid-based mimetic model in assessment of drug-loaded nanoparticles for skin administration.

A lipid-based permeation assay (PVPASC) with a lipid composition similar to Human stratum corneum layer has been previously reported. The aim of this study was to further characterize the PVPASC model in the presence of co-solvents and to determine its applicability to evaluate drug permeability with drug-loaded nanoparticles. Data obtained from PVPASC model were compared with results from isolated SC from pig ear skin. The characterization revealed that the PVPASC barriers retain integrity and calcein permeability when stored up to 12 weeks at -20 °C, in the presence of different co-solvents, and under a skin environment pH range. The permeation profile of calcein in the lipid-based barrier correlated well with data obtained for the isolated SC model and revealed higher reproducibility. Cyclosporine A (CsA) was selected as a model drug, given its relevance for skin-inflammatory diseases and two types of lipid nanoparticles were used to assess the permeability of the PVPASC model. It was possible to distinguish the permeability between free and nanoparticles' loaded cyclosporine. Data obtained with CsA-loaded nanoformulations indicated a higher permeation rate than the obtained for the solid lipid nanoparticles or the free drug. The PVPASC model could be applied as a cost-effective alternative for skin early drug development.

A combined physiological and biophysical approach to understand the ligand-dependent efficiency of 3-hydroxy-4-pyridinone Fe-chelates.

Ligands of the 3-hydroxy-4-pyridinone (3,4-HPO) class were considered eligible to formulate new Fe fertilizers for Iron Deficiency Chlorosis (IDC). Soybean (Glycine max L.) plants grown in hydroponic conditions and supplemented with Fe-chelate [Fe(mpp)3] were significantly greener, had increased biomass, and were able to translocate more iron from the roots to the shoots than those supplemented with an equal amount of the commercially available chelate [FeEDDHA]. To understand the influence of the structure of 3,4-HPO ligand on the role of the Fe-chelate to improve Fe-uptake, we investigated and report here the effect of Fe-chelates ([Fe(mpp)3], [Fe(dmpp)3], and [Fe(etpp)3]) in addressing IDC. Chlorosis development was assessed by measurement of morphological parameters, quantification of chlorophyll and Fe, and other micronutrient contents, as well as measurement of enzymatic activity (FCR) and gene expression (FRO2, IRT1, and Ferritin). All [Fe(3,4-HPO)3] chelates were able to provide Fe to plants and prevent IDC but with a different efficiency depending on the ligand. We hypothesize that this may be related with the distinct physicochemical characteristics of ligands and complexes, namely, the diverse hydrophilic-lipophilic balance of the three chelates. To test the hypothesis, we performed an EPR biophysical study using liposomes prepared from a soybean (Glycine3 max L.) lipid extract and spin probes. The results showed that the most effective chelate [Fe(mpp)3] shows a preferential location close to the surface while the others prefer the hydrophobic region inside the bilayer. SIGNIFICANCE STATEMENT: The 3-hydroxy-4-pyridinone Fe-chelates, [Fe(mpp)3], [Fe(dmpp)3], and [Fe(etpp)3], were all able to provide Fe to plants and prevent IDC. Efficacy is dependent on the structure of the ligand. From an EPR biophysical study using spin probes and liposomes, prepared from a soybean lipid extract, we hypothesize that this may be related with the distinct preferential location close to the surface or on the hydrophobic region of the lipid bilayer. [Fe(mpp)3] provide higher amounts of Fe in the leaves.

Human skin models: From healthy to disease-mimetic systems; characteristics and applications.

Skin drug delivery is an emerging route in drug development, leading to an urgent need to understand the behaviour of active pharmaceutical ingredients within the skin. Given, As one of the body's first natural defences, the barrier properties of skin provide an obstacle to the successful outcome of any skin drug therapy. To elucidate the mechanisms underlying this barrier, reductionist strategies have designed several models with different levels of complexity, using non-biological and biological components. Besides the detail of information and resemblance to human skin in vivo, offered by each in vitro model, the technical and economic efforts involved must also be considered when selecting the most suitable model. This review provides an outline of the commonly used skin models, including healthy and diseased conditions, in-house developed and commercialized models, their advantages and limitations, and an overview of the new trends in skin-engineered models.

Greener and wide applicability range flow-based spectrophotometric method for iron determination in fresh and marine water.

A flow-based method for the spectrophotometric determination of iron in recreational waters, both fresh and marine (variable salinity content), was developed. For that purpose, 3-hydroxy-4-pyrydinone ligand functionalized with an ether function was synthetized and used as chromogenic chelator (1-(3'-methoxypropyl)-2-methyl-3-benzyloxy-4-(1H)pyridinone - MRB13) for iron quantification. This water-soluble reagent was previously reported as a greener alternative to quantify iron, due to its low toxicity and a more environmental friendly synthesis. Furthermore, it also displayed a high affinity and specificity for iron. With the main objective of quantifying iron in a variety of water types (different matrices and iron content), two strategies were developed, one of them including on-line solid-phase extraction (SPE), and the other without resorting to a SPE process. Water matrix clean-up and iron enrichment was achieved using a nitrilotriacetic acid resin column. The potential interference of metal ions usually present in water samples was assessed and no significant interference (<10%) was observed. The limits of detection were 11 and 2.9 µg L-1 without and with SPE, respectively. For one determination (three replicates), the corresponding consumption of MRB13 is 90 µg, sodium hydroxide is 1.4 mg, and boric acid is 5.6 mg. The method was applied to certified water samples and the results were in agreement with certified values. The developed method was also applied to fresh and marine water, and recovery ratios of 103 ± 4 and 101 ± 7 without and with SPE, respectively, were achieved.

Use of an ether-derived 3-hydroxy-4-pyridinone chelator as a new chromogenic reagent in the development of a microfluidic paper-based analytical device for Fe(III) determination in natural waters.

This article reports on the development and validation of a disposable microfluidic paper-based analytical device (µPAD) for on-hand, in-situ, and cheap Fe(III) determination in natural waters complying with World Health Organization guidelines. The developed µPAD used 3-hydroxy-4-pyridinone (3,4-HPO) as a colour reagent due to its considerably lower toxicity than traditionally used iron analytical reagents. It was selected among a group of hydrophilic 3,4-HPO chelators containing ether-derived chains in their structure which were prepared using green methods. The relatively high water solubility of these chelators improved the detection limit and applicability as µPAD reagents. Under optimal conditions, the µPAD is characterised by a quantification range between 0.25 and 2.0 mg/L, a detection limit of 55 µg/L and 15 min of analysis time. The signal stability extends up to 4 h and the device is stable for at least one month. The reagent consumption is below 0.2 mg per analysis and the µPAD method was validated by analysis certified reference materials and by comparison with atomic absorption results (RD < 10%). The newly developed µPAD was successfully applied to the determination of iron in river, well and tap waters with no need of any prior sample pre-treatment.

Tuning the Anti(myco)bacterial Activity of 3-Hydroxy-4-pyridinone Chelators through Fluorophores.

Controlling the sources of Fe available to pathogens is one of the possible strategies that can be successfully used by novel antibacterial drugs. We focused our interest on the design of chelators to address Mycobacterium avium infections. Taking into account the molecular structure of mycobacterial siderophores and considering that new chelators must be able to compete for Fe(III), we selected ligands of the 3-hydroxy-4-pyridinone class to achieve our purpose. After choosing the type of chelating unit it was also our objective to design chelators that could be monitored inside the cell and for that reason we designed chelators that could be functionalized with fluorophores. We didn't realize at the time that the incorporation a fluorophore, to allow spectroscopic detection, would be so relevant for the antimycobacterial effect or to determine the affinity of the chelators towards biological membranes. From a biophysical perspective, this is a fascinating illustration of the fact that functionalization of a molecule with a particular label may lead to a change in its membrane permeation properties and result in a dramatic change in biological activity. For that reason we believe it is interesting to give a critical account of our entire work in this area and justify the statement "to label means to change". New perspectives regarding combined therapeutic approaches and the use of rhodamine B conjugates to target closely related problems such as bacterial resistance and biofilm production are also discussed.

New fluorescent rosamine chelator showing promising antibacterial activity against Gram-positive bacteria.

The restricted number of antibiotics to treat infections caused by common multidrug resistant bacterial pathogens in the clinical setting demands a continuous search for new molecules with antibacterial properties. Bacterial iron deprivation represents a promising alternative, being iron chelators an attractive class for drug design in which particular compounds seem to have antibacterial effect. In this work, we report the synthesis and characterization of a new fluorescent 3-hydroxy-4-pyridinone (3,4-HPO) iron chelator functionalized with a carboxyrosamine fluorophore (MRB20). The antibacterial activity of MRB20 was assessed against representative strains from clinically relevant Gram-positive and Gram-negative bacterial species and further compared with the inhibitory effect of a set of structurally related iron chelators including Deferiprone (1,2-dimethyl-3-hydroxy-4-pyridinone). Compounds exhibiting a promising minimal inhibitory concentration (MIC < 10 mg/L) were further tested against a wider range of bacterial genera and species (Staphylococcus spp. Enterococcus spp. Listeria monocytogenes, Bacillus spp.), including multidrug resistant bacteria. With the exception of the novel compound (MRB20), all chelators inhibited the strains assayed at very high concentrations [minimum inhibitory concentrations (MIC) ranging from 70 mg/L to >180 mg/L]. MRB20 revealed a good antibacterial activity (6.7-13.2 mg/L) against Gram-positive strains from different genera and species, including clinically relevant species (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis), which might be eventually compatible with a therapeutic application or as adjuvant.

Insights on the relationship between structure vs. toxicological activity of antibacterial rhodamine-labelled 3-hydroxy-4-pyridinone iron(III) chelators in HepG2 cells.

In the present study we investigated the in vitro hepatotoxicity of a set of rhodamine-labelled 3-hydroxy-4-pyridinones (3,4-HPO) that had previously demonstrated significant inhibitory effect in the intramacrophagic growth of Mycobacterium avium. Our aim was to establish a correspondence between the molecular structure and the in vitro toxicological activity of these compounds. The impact of a set of bidentate (MRB2, MRB7, MRB8, and MRB9) and hexadentate (MRH7, MRH8, and MRH10) chelators on cellular metabolic competence and membrane integrity was investigated in HepG2 cells. Our findings indicate that: a) hexadentate chelators are more cytotoxic than parent bidentate ligands; b) disruption of cell membrane and metabolic competence only occurred after 5 days, at the highest concentrations tested; c) strict correlation between bacteriostatic activity and in vitro toxicity was observed, which seems to be directly dependent on the size of the molecule and on the hydrophilic/lipophilic balance; d) among the set of bidentate ligands, carboxyrhodamine derivatives (amide linker) presented lower detrimental effects, when compared with rhodamine B isothiocyanate chelators (thiourea linker); e) contrarily, for the hexadentate series, rhodamine B isothiocyanate derivatives are less cytotoxic to HepG2 cells than carboxyrhodamine molecules; and f) for all compounds tested, when the substituents of the nitrogen atom were switched from ethyl to methyl, an increment of toxicity was observed. Overall, all chelators seem to display suitable in vitro toxicological potential to combat fast grow bacteria. According to their in vitro pharmacological: toxicological potential ratio, MRH7 and MRH8 may be considered as the most suitable compounds to undergo further pre-clinical development studies.

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium.

Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17-30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17-30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17-30 did not localize to M. avium-harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17-30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis, M. leprae, M. avium, etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we show that peptides derived from bovine lactoferricin (LFcin) improve the antimicrobial activity of ethambutol against Mycobacterium avium growing inside macrophages. Moreover, the d-enantiomer of a short version of lactoferricin containing amino acids 17 to 30 (d-LFcin17-30) causes intramacrophagic death of M. avium by increasing the formation of lysosomes and autophagosomes. This work opens the way to the use of lactoferricin-derived peptides to treat infections caused by mycobacteria and highlights important modulatory effects of d-FLcin17-30 on macrophages, which may be useful under other conditions in which macrophage activation is needed.