Peptide-functionalized chitosan-based microcapsules for dual active targeted treatment of lung infections.

Lung infections, such as: pneumonia, chronic obstructive cystic fibrosis, tuberculosis are generally caused by viruses, bacteria and fungi. As these infections are very difficult to treat, new therapeutic approaches are investigated in order to maximize the efficiency of the treatment and to reduce the major complications that can occur. The main objective of this study was focused on the preparation of drug-loaded peptides-functionalized microcapsules, obtained by a double emulsion, based on carboxylated chitosan (CMCS), poly(vinyl alcohol) (PVA) and an activator [4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride] (DMT-MM), for the dual active targeting and treatment of pulmonary infections. The microcapsules were functionalized on the surface with both CGSPGWVRC and indolicidin (IN) peptides, as effective ligands for the active targeting of both alveolar capillary endothelial cells and bacterial cells. FTIR spectroscopy confirmed the formation of ester and amide bonds into the structure of prepared microcapsules. Microcapsules diameter varied between 893 and 965 nm. The swelling degree in PBS, at pH 7.4, ranged between 1760 %- 2100 %. All the analyzed samples showed hemolysis degrees lower than 2 %, which demonstrated their non-hemolytic character. Evaluation of the impact of microcapsules on WI-38 normal human lung cells and RAW 264.7 mouse macrophages revealed a non-toxic or slightly cytotoxic effect. Internalization assay proved that microcapsules were localized at intracellular level.

[RAST, Inmunoblot, Immunocap and ISAC in food allergy]. / RAST, Inmunoblot, Inmunocap e ISAC en alergia alimentaria.

skin tests; Sensitization; IgE-mediated allergy; allergenic extract. After the discovery of IgE, technological advances have provided new laboratory tools for the quantification of allergen-specific IgE antibodies in serum and on the surface of basophils-mast cells. In vitro testing offers numerous advantages: accurate quantitation, lack of drug interference, safety, and long-term storage of samples. Quantitative immunoassays for IgE antibodies can be an adjunct to skin testing. The allergen reagent in solid phase (allergosorbent) or liquid is the main component of the assay that confers specificity to the IgE antibody test. It is the most complex and highly variable reagent in IgE antibody assays. The choice to use diagnostic recombinants on a single rather than multiple platforms is made on a case-by-case basis (considering prior history and clinical profile) and in an allergen-dependent manner. Although most food allergies are limited to a small number of possible triggers, these foods are very complex when evaluating their allergenic potential. The possibility of fractionating the allergen and understanding some of its components as potentially important to define the risk of clinical reaction, cross-reactivity, or persistence of allergy, opened a new era in the field of allergy, called molecular allergy. The identification of the allergenic component responsible for the reactions is an important tool to confirm the information and severity of the symptoms, natural history of the disease, possibility of cross-reactivity and clinical symptoms (allergy markers).

Después del descubrimiento de la IgE, los avances tecnológicos han proporcionado nuevas herramientas de laboratorio para la cuantificación de anticuerpos IgE específicos de alérgenos en suero y en la superficie de basófilos-mastocitos. Las pruebas in vitro ofrecen numerosas ventajas: cuantificación precisa, falta de interferencia de fármacos, seguridad y almacenamiento a largo plazo de las muestras. Los inmunoensayos cuantitativos para anticuerpos IgE pueden ser un complemento de las pruebas cutáneas. El reactivo de alergeno en fase sólida (alergosorbente) o líquida es el componente principal del ensayo que confiere especificidad a la prueba de anticuerpos IgE. Es el reactivo más complejo y altamente variable en los ensayos de anticuerpos IgE. La elección de utilizar recombinantes de diagnóstico en una única plataforma en lugar de múltiples se realiza caso por caso (considerando el historial previo y el perfil clínico) y de manera dependiente de los alérgenos. Aunque la mayor parte de las alergias alimentarias se limitan a una pequeña cantidad de posibles desencadenantes, estos alimentos son muy complejos al momento de evaluar su potencial alergénico. La posibilidad de fraccionar el alérgeno y entender algunos de sus componentes como potencialmente importantes para definir el riesgo de reacción clínica, reactividad cruzada o persistencia de la alergia, abrió una nueva era en el campo de la alergia, denominada alergia molecular. La identificación del componente alergénico responsable de las reacciones supone una herramienta importante para confirmar la información y gravedad de los síntomas, historia natural de la enfermedad, posibilidad de reactividad cruzada y clínica (marcadores de alergia).

Whey proteins as multifunctional food materials: Recent advancements in hydrolysis, separation, and peptidomimetic approaches.

Whey protein derived bioactives, including α-lactalbumin, ß-lactoglobulin, bovine serum albumin, lactoferrin, transferrin, and proteose-peptones, have exhibited wide ranges of functional, biological and therapeutic properties varying from anticancer, antihypertensive, and antimicrobial effects. In addition, their functional properties involve gelling, emulsifying, and foaming abilities. For these reasons, this review article is framed to understand the relationship existed in between those compound levels and structures with their main functional, biological, and therapeutic properties exhibited either in vitro or in vivo. The impacts of hydrolysis mechanism and separation techniques in enhancing those properties are likewise discussed. Furthermore, special emphasize is given to multifunctional effects of whey derived bioactives and their future trends in ameliorating further food, pharmaceutical, and nutraceutical products. The underlying mechanism effects of those properties are still remained unclear in terms of activity levels, efficacy, and targeted effectiveness. For these reasons, some important models linking to functional properties, thermal properties and cell circumstances are established. Moreover, the coexistence of radical trapping groups, chelating groups, sulfhydryl groups, inhibitory groups, and peptide bonds seemed to be the key elements in triggering those functions and properties. Practical Application: Whey proteins are the byproducts of cheese processing and usually the exploitation of these food waste products has increasingly getting acceptance in many countries, especially European countries. Whey proteins share comparable nutritive values to milk products, particularly on their richness on important proteins that can serve immune protection, structural, and energetic roles. The nutritive profile of whey proteins shows diverse type of bioactive molecules like α-lactalbumin, ß-lactoglobulin, lactoferrin, transferrin, immunoglobulin, and proteose peptones with wide biological importance to the living system, such as in maintaining immunological, neuronal, and signaling roles. The diversification of proteins of whey products prompted scientists to exploit the real mechanisms behind of their biological and therapeutic effects, especially in declining the risk of cancer, tumor, and further complications like diabetes type 2 and hypertension risk effects. For these reasons, profiling these types of proteins using different proteomic and peptidomic approaches helps in determining their biological and therapeutic targets along with their release into gastrointestinal tract conditions and their bioavailabilities into portal circulation, tissue, and organs. The wide applicability of those protein fractions and their derivative bioactive products showed significant impacts in the field of emulsion and double emulsion stabilization by playing roles as emulsifying, surfactant, stabilizing, and foaming agents. Their amphoteric properties helped them to act as excellent encapsulating agents, particularly as vehicle for delivering important vitamins and bioactive compounds. The presence of ferric elements increased their transportation to several metal-ions in the same time increased their scavenging effects to metal-transition and peroxidation of lipids. Their richness with almost essential and nonessential amino acids makes them as selective microbial starters, in addition their richness in sulfhydryl amino acids allowed them to act a cross-linker in conjugating further biomolecules. For instance, conjugating gold-nanoparticles and fluorescent materials in targeting diseases like cancer and tumors in vivo is considered the cutting-edges strategies for these versatile molecules due to their active diffusion across-cell membrane and the presence of specific transporters to these therapeutic molecules.

The Study on Timolol and Its Potential Phototoxicity Using Chemical, In Silico and In Vitro Methods.

Timolol (TIM) is a non-selective ß-adrenergic receptor antagonist used orally for the treatment of hypertension and heart attacks, and topically for treating glaucoma; lately, it has also been used in some specific dermatological problems. In the present study, its photodegradation and potential risk of phototoxicity were examined using chemical, in silico and in vitro methods. The UV/VIS irradiated solutions of TIM at pH 1-13 were subjected to LC-UV and UPLC-HRMS/MS analyses showing pseudo first-order kinetics of degradation and several degradation products. The structures of these photodegradants were elucidated by fragmentation path analysis based on high resolution (HR) fragmentation mass spectra, and then used for toxicity evaluation using OSIRIS Property Explorer and Toxtree. Potential risk of phototoxicity was also studied using chemical tests for detecting ROS under UV/VIS irradiation and in vitro tests on BALB/c 3T3 mouse fibroblasts (MTT, NRU and Live/Dead tests). TIM was shown to be potentially phototoxic because of its UV/VIS absorptive properties and generation ROS during irradiation. As was observed in the MTT and NRU tests, the co-treatment of fibroblasts with TIM and UV/VIS light inhibited cell viability, especially when concentrations of the drug were higher than 50 µg/mL.

Influence of ZnO Nanoparticles on the Properties of Ibuprofen-Loaded Alginate-Based Biocomposite Hydrogels with Potential Antimicrobial and Anti-Inflammatory Effects.

Hydrogels are a favorable alternative to accelerate the burn wound healing process and skin regeneration owing to their capability of absorbing contaminated exudates. The bacterial infections that occur in burn wounds might be treated using different topically applied materials, but bacterial resistance to antibiotics has become a major problem worldwide. Therefore, the use of non-antibiotic treatments represents a major interest in current research. In this study, new antibiocomposite hydrogels with anti-inflammatory and antimicrobial properties based on hyaluronic acid (HA) and sodium alginate (AG) were obtained using 4-(4,6-dimethoxy-1,3,5-triazinyl-2)-4-methylmorpholinium chloride as an activator. The combination of Ibuprofen, a non-steroidal anti-inflammatory drug commonly used to reduce inflammation, fever and pain in the body, with zinc oxide nanoparticles (ZnO NPs) was used in this study aimed at creating a complex hydrogel with anti-inflammatory and antimicrobial action and capable of improving the healing process of wounds caused by burns. FTIR spectra confirmed the cross-linking of AG with HA as well as the successful incorporation of ZnO NPs. Using electronic microscopy, it was noticed that the morphology of hydrogels is influenced by the incorporation of ZnO nanoparticles. Moreover, the incorporation of ZnO nanoparticles into hydrogels also has an influence on the swelling behavior at both pH 7.4 and 5.4. In fact, the swelling rate is lower when the amounts of the activator, HA and ZnO NPs are high. A drug release rate of almost 100% was observed for hydrogels without ZnO NPs, whereas the addition of nanoparticles to hydrogels led to a decrease in the release rate to 68% during 24 h. Cellular viability tests demonstrated the non-cytotoxic behavior of the hydrogels without the ZnO NPs, whereas a weak to moderate cytotoxic effect was noticed for hydrogels with ZnO NPs. The hydrogels containing 4% and 5% ZnO NPs, respectively, showed good antimicrobial activity against the S. aureus strain. These preliminary data prove that these types of hydrogels can be of interest as biomaterials for the treatment of burn wounds.

The influence of Leucidal - eco-preservative from radish - on model lipid membranes and selected pathogenic bacteria.

In this work the effect of Leucidal - a natural preservative from radish dedicated to be used in cosmetics - on bacteria cells and model bacteria membranes was investigated. To get insight into the mechanism of action of this formulation the lipid Langmuir monolayers imitating Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) membranes were prepared. Then, the influence of Leucidal on model systems was investigated by means of the surface pressure/area measurements, penetration studies and Brewster Angle Microscopy (BAM) visualization. Similar experiments were done also for one component monolayers formed from the model membrane lipids. The in vitro tests were done on five different bacteria species (E. coli, Enterococcus faecalis, S. aureus, Salmonella enterica, Pseudomonas aeruginosa). Leucidal was found to decrease packing of the monolayers, however, it was excluded from the films at higher concentrations. Model membrane experiments evidenced also a stronger affinity of the components of this eco-preservative to E. coli vs S. aureus membrane. Among one component films, those formed from phosphatidylglycerols and cardiolipins were more sensitive to the presence of Leucidal. However, in vitro tests evidenced that Leucidal exerts stronger inhibitory effect against S. aureus bacteria as compared to E. coli strain. These findings were discussed from the point of view of the role of Leucidal components and the lipid membrane properties in the membrane - based mechanism of action of this preservative. The results allow one to suggest that the membrane may not be the main site of action of Leucidal on bacteria. Moreover, since high concentration of the tested preparation exerted antibacterial activity in relation to all tested bacteria, a low selectivity of Leucidal can be postulated, which may be problematic from the point of view of its effect on the skin microbiome.

Comparison of Skin Prick Tests (SPT), Intradermal Tests (IDT) and In Vitro Tests in the Characterization of Insect Bite Hypersensitivity (IBH) in a Population of Lusitano Horses: Contribution for Future Implementation of SPT in IBH Diagnosis.

Thirty controls (C) and 30 IBH-affected (T) Lusitano horses were evaluated. T horses were included based on anamnesis and physical examination, supported by questionnaires. All horses were submitted to skin tests, Intrademal (IDT) and Skin Prick Tests (SPT), on the neck with 14 specific allergens, 13 recombinant proteins (r-proteins) from Culicoides nubeculosus (Cul n) and Culicoides obsoletus (Cul o) salivary glands and Culicoides nubeculosus Whole Body Extract (Cul n WBE). Addicionally, a cluster of six T and six C horses were also tested with Cul n 3 and Cul n 4 produced in insect cells and barley, as well as E. coli produced Cul o 3 and Cul o WBE. Allergen concentrations were 10 µg/mL for IDT and 100 µg/mL for SPT, and wheal diameters assessed at 20 min, 6 and 48 h. IDTs were considered positive when wheal diameter was ≥50% of the histamine wheal and SPT's ≥ 0.9 cm. In vitro tests, allergen-specific serum IgE and sulfidoleukotriene (sLT) release assay were also carried out. Results showed that Cul n WBE, Cul n 7, 8, 9, Cul o1P and Cul o 2P were the best performing allergens for SPTs (p ≤ 0.0001) for the 1st allergen panel and Cul o WBE, Cul n 3 Bar and Cul n 4 Bac (p ≤ 0.05) for the 2nd, presenting a higher discriminatory diagnostic potential than IDTs, at a concentration of 100 µg/mL, with readings assessed at 20 min. Regarding in vitro tests overall, the sLT release assay performed best.

Study on the thyroid function of male and female thoroughbred horses in different times after winning races at the hippodrome Cidade Jardim, with the use of in vitro 125I-T4 and 125l-T3 tests / Estudo da função tiróides em equinos machos e fêmeas Puro Sangue Inglês, em diferentes tempos, após terem vencido carreira no Hipódromo de Cidade Jardim, mediante a utilização dos testes in vitro 125I-T3 e 125I-T4

The author has studied nine animals, five females and four males, observing also data on age, weight, pulse, breathing and temperature taken before and after the race, besides the values of T3 and T4 Tests of the sera collected 30, 60, 90 and 120 minutes after the event. We have performed variance and regression analysis for the results of T3 and T4 Tests obtained from this group. For T3 Test significance was found as to sex, along with linear regression, with the same results for T4 Test without linear regression. We have added the data relative to T3 Test of this group with those obtained from sera of male and female horses, in repose and after activity, to the values obtained from females over 90 days pregnant verifying statistical significance through variance analysis. With the help of Tukeys test, we have noticed, in this case, differences among the three groups. The data referring to T4 Test of sera of male and female horses after winning a race, in repose, after activity, with data obtained from females over 90 days pregnant showed statistical significance by variance analysis. Using Tukeys test in the four groups, we found that only the horses in repose group differed from the others.

Foram estudados 9 animais, sendo 5 fêmeas e 4 machos, onde se observou, também, dados sobre idade, peso, pulso, respiração e temperatura, tomadas antes e depois da corrida, além dos valores dos Testes T3 e T4 dos soros colhidos, 30, 60, 90 e 120 minutos após a prova. Foram feitas análises de variância e de regressão para os resultados dos Testes T3 e T4 obtidos neste lote. Para o Teste T3 houve significância quanto ao sexo observando-se, também, regressão linear, obtendo-se o mesmo para o Teste T4, sem regressão linear. Confrontamos os dados referentes ao Teste T3 deste lote com os conseguidos de soros de equinos, machos e fêmeas, em descanso e em trabalho, com os valores obtidos em fêmeas em gestação acima de 90 dias, verificando-se significância estatística mediante a análise da variância. Aplicando-se o Teste de Tukey, evidenciamos, neste caso, que os 3 lotes diferiam entre si. Os dados referentes ao Teste T4, de soros de equinos, machos e fêmeas, após terem vencido corrida, em descanso, em trabalho, com dados obtidos de fêmeas em gestação acima de 90 dias, verificando-se à custa da análise da variância, significância estatística. Aplicando-se o teste de Tukey aos 4 lotes, observou-se que apenas o lote dos equinos em descanso diferiu dos demais.

Study of the thyroid function in dogs, with the use of "in vitro I-T 3 and 125 I-T4 tests / Estudo da função tiróidea em cães, mediante a utilização de testes IN VITRO 125 l-T3 e 125 I-T4

Using Res-O-Mat T 3 Micro test liothyronine I125 e Res-O-Mat T4 I125, a research has been made about the thyroid function on 60 dogs, clinically normal on various races, males and females divided due to the weight body, been the first group made of animals between 1 to 7 kg, the second from 7 to 15 and the last one of dogs which weigh are more than 15 kg. It has been found for the 125I-T3. Test, the average 41,60 (s=1,58); 41,33(s=2,20) e 4 1,70(s=1,85), respectively for the first, second and third groups, obtaining as an average 41,54% of retention in resin. For the 125I-T4 test it has been found the average 1,76 (s=0,57); 2,01 (s=0,74) and 1,94 (s=0,60), respectively for the first, second and third groups, being 1,90 mcg/1OOml of serum the average for the three groups. It has not been observed statistics significance with reference to sex, age and size.

Utilizando Res-o-Mat T 3 Micro Test Liothyronine I125 e Res-O-Mat T4 I125 realizamos pesquisa sobre o estado funcional tiróideo de 60 cães clinicamente normais, de diversas raças, machos e fêmeas, divididos segundo o peso corpóreo, sendo o primeiro grupo constituído de animais entre 1 a 7 kg, o segundo de 7 a 15 kg e o último de cães acima de 15 kg de peso. Encontramos para os testes 125I-T3, as médias 41,60 (S:1,58); 41,33 (S:2,20) e 41,70 (S:1,85), respectivamente para os lotes de pequeno, médio e grande porte, obtendo-se para os três lotes a média de 41,54% de retenção em resina. Para os testes 125I-T4, obtivemos as médias 1,76 (S:0,57); 2,01 (S:0,74) e 1,94 (S:0,60), respectivamente para os lotes de pequeno, médio e grande porte, sendo 1,90 mcg/1OOml de soro a média para os três grupos. Não foi observada significância estatística com referência ao sexo, idade e porte.

Tools for Etiologic Diagnosis of Drug-Induced Allergic Conditions.

Drug hypersensitivity reactions are a serious concern in clinical practice because they can be severe and result in lifelong sequelae. An accurate diagnosis and identification of the culprit drug is essential to prevent future reactions as well as for the identification of safe treatment alternatives. Nonetheless, the diagnosis can be challenging. In vivo and in vitro tests can be helpful, although none are conclusive; therefore, the tests are not usually performed in isolation but as part of a diagnostic algorithm. In addition, some in vitro tests are only available in research laboratories, and standardization has not been fully accomplished. Collaborating research is needed to improve drug hypersensitivity reaction diagnosis. In this review, we update the current available in vivo and in vitro tools with their pros and cons and propose an algorithm to integrate them into clinical practice.

Which value aspects are relevant for the evaluation of medical devices? Exploring stakeholders' views through a Web-Delphi process.

BACKGROUND: Implementation and uptake of health technology assessment for evaluating medical devices require including aspects that different stakeholders consider relevant, beyond cost and effectiveness. However, the involvement of stakeholders in sharing their views still needs to be improved. OBJECTIVE: This article explores the relevance of distinct value aspects for evaluating different types of medical devices according to stakeholders' views. METHODS: Thirty-four value aspects collected through literature review and expert validation were the input for a 2-round Web-Delphi process. In the Web-Delphi, a panel of participants from five stakeholders' groups (healthcare professionals, buyers and policymakers, academics, industry, and patients and citizens) judged the relevance of each aspect, by assigning a relevance-level ('Critical', 'Fundamental', 'Complementary', or 'Irrelevant'), for two types of medical devices separately: 'Implantable' and 'In vitro tests based on biomarkers'. Opinions were analysed at the panel and group level, and similarities across devices were identified. RESULTS: One hundred thirty-four participants completed the process. No aspects were considered 'Irrelevant', neither for the panel nor for stakeholder groups, in both types of devices. The panel considered effectiveness and safety-related aspects 'Critical' (e.g., 'Adverse events for the patient'), and costs-related aspects 'Fundamental' (e.g., 'Cost of the medical device'). Several additional aspects not included in existing frameworks' literature, e.g., related to environmental impact and devices' usage by the healthcare professional, were deemed as relevant by the panel. A moderate to substantial agreement across and within groups was observed. CONCLUSION: Different stakeholders agree on the relevance of including multiple aspects in medical devices' evaluation. This study produces key information to inform the development of frameworks for valuing medical devices, and to guide evidence collection.

In vitro diagnostic testing for drug allergy in children.

Diagnosing Drug Hypersensitivity Reactions (DHRs) could be a complicated process especially in children, since allergic-like manifestation at this age is more often the expression of concomitant infections rather than a actual DHRs. In vivo tests are usually suggested as a first step; however, prick and intradermal tests could be painful and have shown different sensitivity and specificity among published studies. In some cases, in vivo tests such as Drug Provocation test (DPT) could be even contraindicated. Therefore, the need for in vitro testing is compelling, to add useful information along the diagnostic pathway and to limit the need of DPT. In this review, we analyze the different types of in vitro tests, focusing on those used more widely such as specific IgE and on those that are still for research settings, such as basophil activation test and lymphocyte transformation test, but that have shown some useful diagnostic potential.

Evaluation of in vitro toxicity information for zebrafish as a promising alternative for chemical hazard and risk assessment.

In vitro assays are widely proposed as a test alternative to traditional in vivo standard acute and chronic toxicity tests. However, whether toxicity information derived from in vitro assays instead of in vivo tests could provide sufficient protection (e.g., 95 % of protection) for chemical risks remain evaluated. To investigate the feasibility of zebrafish (Danio rerio) cell-based in vitro test method as a test alternative, we comprehensively compared sensitivity differences among endpoints, among test methods (in vitro, FET and in vivo), and between zebrafish and rat (Rattus norvegicus), respectively using chemical toxicity distribution (CTD) approach. For each test method involved, sublethal endpoints were more sensitive than lethal endpoints for both zebrafish and rat, respectively. Biochemistry (zebrafish in vitro), development (zebrafish in vivo and FET), physiology (rat in vitro) and development (rat in vivo) were the most sensitive endpoints for each test method. Nonetheless, zebrafish FET test was the least sensitive one compared to its in vivo and in vitro tests for either lethal or sublethal responses. Comparatively, rat in vitro tests considering cell viability and physiology endpoints were more sensitive than rat in vivo test. Zebrafish was found to be more sensitive than rat regardless of in vivo or in vitro tests for each pairwise endpoint of concern. Those findings indicate that zebrafish in vitro test is a feasible test alternative to zebrafish in vivo and FET test and traditional mammalian test. It is suggesting that zebrafish in vitro test can be optimized by choosing more sensitive endpoints, such as biochemistry to provide sufficient protection for zebrafish in vivo test and to establish applications of zebrafish in vitro test in future risk assessment. Our findings are vital for evaluating and further application of in vitro toxicity toxicity information as an alternative for chemical hazard and risk assessment.

Synthesis and evaluation of anti-yellow fever virus activity of new 6-aryl-3-R-amino-1,2,4-triazin-5(4H)-ones.

Yellow fever disease is one of public health concerns in the tropics. Despite its significant medicinal and economic impact among large groups of the population, there is a lack of effective treatment against yellow fever. In this regard, here we describe the synthesis of a series of new 6-aryl-3-R-amino-1,2,4-triazin-5(4H)-ones and evaluation of their in vitro inhibitory activity against yellow fever virus. Among all tested compounds 4 derivatives possessing strong inhibitory activity at µM concentrations were identified. All the active compounds revealed a good toxicity profile. These facts make the compounds interesting candidates for further evaluation of their efficacy in the treatment of yellow fever virus infection in vivo.

Modeling of Biological Activity of PEO-Coated Titanium Implants with Conjugates of Cyclic RGD Peptide with Amino Acid Bisphosphonates.

Titanium is considered to be the most essential metal in the field of implantology. The main factors determining metal biocompatibility, among others, include the morphology and chemical composition of the titanium surface. Therefore, the aim of this work was to develop approaches to control the biological activity of the titanium surface by creating coatings that combine both an inorganic phase with a given morphology and organic molecules containing an integrin-selective peptide that regulate cell adhesion and proliferation. As such, we synthesized new c(RGDfC) derivatives of amino acid bisphosphonates (four examples) with different bisphosphonate anchors and maleimide linkers. These molecules were deposited on a highly developed porous surface obtained via the plasma electrolytic oxidation (PEO) of coarse-grained and nanostructured titanium. In vitro studies demonstrated the increase in the viability degree of mesenchymal stem cells and fibroblasts on the surface of coarse-grained or nanostructured titanium modified with PEO and a c(RGDfC) derivative of ε-aminocaproic acid bisphophonate with an SMCC linker. As a result, the use of conjugates of amino acid bisphosphonates with a cyclic RGD peptide for the modification of PEO-coated titanium opens the ways for the effective control of the biological activity of the metal implant surface.

Assessment of the Actual Toxicity of Engine Exhaust Gas Emissions from Euro 3 and Euro 6 Compliant Vehicles with the BAT-CELL Method Using In Vitro Tests.

Legal restrictions on vehicle engine exhaust gas emission control do not always go hand in hand with an actual reduction in the emissions of toxins into the atmosphere. Moreover, the methods currently used to measure exhaust gas emissions do not give unambiguous results on the impact of the tested gases on living organisms. The method used to assess the actual toxicity of gases, BAT-CELL Bio-Ambient-Tests using in vitro tests, takes into account synergistic interactions of individual components of a mixture of gases without the need to know its qualitative and quantitative composition and allows for determination of the actual toxicity of the gas composition. Using the BAT-CELL method, exhaust gases from passenger vehicles equipped with spark-ignition engines complying with the Euro 3 and Euro 6 emission standards were tested. The results of toxicological tests were correlated with the results of chromatographic analysis. It was shown that diverse qualitative composition of the mixture of hydrocarbons determining the exhaust gases toxicity may decrease the percentage value of cell survival. Additionally, it was proven that the average survival of cells after exposure to exhaust gases from tested vehicles meeting the more restrictive Euro 6 standard was lower than for vehicles meeting the Euro 3 standard thus indicating the higher toxicity of exhaust gases from newer vehicles.

Holm Oak (Quercus ilex subsp. ballota (Desf.) Samp.) Bark Aqueous Ammonia Extract for the Control of Invasive Forest Pathogens.

Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) bark is a commonly used remedy to treat gastrointestinal disorders, throat and skin infections, hemorrhages, and dysentery. It has also been previously reported that its methanol extracts possess antibacterial activity, which can be related to the richness of Quercus spp. extracts in phenolic compounds, such as flavonoids and tannins. However, there is no information on the antifungal (including oomycete) properties of the bark from Q. ilex or its subspecies (ilex and ballota). In this work, we report the characterization of the aqueous ammonia extract of its bark by FTIR and GC-MS and the results of in vitro and ex situ inhibition tests against three phytopathogens. The main phytochemical components identified were inositols (19.5%), trans-squalene (13%), 4-butoxy-1-butanol (11.4%), gulopyranose (9.6%), lyxose (6.5%), 2,4-dimethyl-benzo[H]quinoline (5.1%), catechol (4.5%), and methoxyphenols (4.2%). The efficacy of the extract in controlling forest phytopathogens was tested in vitro against Fusarium circinatum (responsible for pitch canker of Pinus spp.), Cryphonectria parasitica (which causes chestnut blight), and Phytophthora cinnamomi (which causes 'root and crown rot' in a variety of hosts, including Castanea, conifers, Eucalyptus, Fagus, Juglans, Quercus, etc.), obtaining EC90 values of 322, 295, and 75 µg·mL-1, respectively, much lower than those attained for a commercial strobilurin fungicide (azoxystrobin). The extract was further tested ex situ against P. cinnamomi on artificially inoculated, excised stems of 'Garnem' almond rootstock, attaining complete protection at a dose of 782 µg·mL-1. The results suggest that holm oak bark extract may be a promising source of bioactive compounds against invasive forest pathogens, including the oomycete that is causing its decline, the so-called 'seca' in Spain.

Structuring and De-Structuring of Nanovectors from Algal Lipids: Simulated Digestion, Preliminary Antioxidant Capacity and In Vitro Tests.

Biocompatible nanocarriers can be obtained by lipid extraction from natural sources such as algal biomasses, which accumulate different lipid classes depending on the employed culture media. Lipid aggregates can be distinguished according to supramolecular architecture into lamellar and nonlamellar structures. This distinction is mainly influenced by the lipid class and molecular packing parameter, which determine the possible values of interfacial curvature and thus the supramolecular symmetries that can be obtained. The nanosystems prepared from bio-sources are able to self-assemble into different compartmentalized structures due to their complex composition. They also present the advantage of increased carrier-target biocompatibility and are suitable to encapsulate and vehiculate poorly water-soluble compounds, e.g., natural antioxidants. Their functional properties stem from the interplay of several parameters. Following previous work, here the functionality of two series of structurally distinct lipid nanocarriers, namely liposomes and cubosomes deriving from algal biomasses with different lipid composition, is characterized. In the view of their possible use as pharmaceutical or nutraceutical formulations, both types of nanovectors were loaded with three well-known antioxidants, i.e., curcumin, α-tocopherol and piperine, and their carrier efficacy was compared considering their different structures. Firstly, carrier stability in biorelevant conditions was assessed by simulating a gastrointestinal tract model. Then, by using an integrated chemical and pharmacological approach, the functionality in terms of encapsulation efficiency, cargo bioaccessibility and kinetics of antioxidant capacity by UV-Visible spectroscopy was evaluated. Subsequently, in vitro cytotoxicity and viability tests after administration to model cell lines were performed. As a consequence of this investigation, it is possible to conclude that nanovectors from algal lipids, i.e., cubosomes and liposomes, can be efficient delivery agents for lipophilic antioxidants, being able to preserve and enhance their activity toward different targets while promoting sustained release.

Osteoblastic cell response to Al2O3-Ti composites as bone implant materials.

Introduction: Alumina-titanium (Al2O3-Ti) composites with enhanced mechanical and corrosion properties have been recently developed for potential applications in orthopaedics and hard tissue replacements. However, before any clinical use, their interactions with biological environment must be examined. Methods: The aim of this study, therefore, was to assess the biocompatibility of three Al2O3-Ti composites having 25, 50, and 75 volume percentages of titanium. These materials were made by spark plasma sintering (SPS), and MC3T3-E1 cells were cultured onto the sample discs to evaluate the cell viability, proliferation, differentiation, mineralization, and adhesion. Furthermore, the apatite formation ability and wettability of the composites were analysed. Pure Ti (100Ti) and monolithic Al2O3 (0Ti) were also fabricated by SPS and biological characteristics of the composites were compared with them. Results: The results showed that cell viability to 75Ti (95.0%), 50Ti (87.3%), and 25Ti (63.9%) was superior when compared with 100Ti (42.7%). Pure Al2O3 also caused very high cell viability (89.9%). Furthermore, high cell proliferation was seen at early stage for 50Ti, while the cells exposed to 75Ti proliferated more at late stages. Cell differentiation was approximately equal between different groups, and increased by time. Matrix mineralization was higher on the composite surfaces rather than on 0Ti and 100Ti. Moreover, the cells adhered differently to the surfaces of different biomaterials where more spindle-shaped configuration was found on 100Ti, slightly enlarged cells with dendritic shape and early pseudopodia were observed on 75Ti, and more enlarged cells with long dendritic extensions were found on 0Ti, 25Ti, and 50Ti. The results of EDS analysis showed that both Ca and P deposited on the surfaces of all materials, after 20 days of immersion in SBF. Conclusion: Our in-vitro findings demonstrated that the 75Ti, 50Ti, and 25Ti composites have high potential to be used as load-bearing orthopedic materials.

Trends in the Design and Evaluation of Polymeric Nanocarriers: The In Vitro Nano-Bio Interactions.

Different types of natural and synthetic polymeric nanocarriers are being tested for diverse biomedical applications ranging from drug/gene delivery vehicles to imaging probes. The development of such innovative nanoparticulate systems (NPs) should include in the very beginning of their conception a comprehensive evaluation of the nano-bio interactions. Specifically, intrinsic physicochemical properties as size, surface charge and shape may have an impact on cellular uptake, intracellular trafficking, exocytosis and cyto- or genocompatibility. Those properties can be tuned for effectiveness purposes such as targeting intracellular organelles, but at the same time inducing unforeseen adverse nanotoxicological effects. Further, those properties may change due to the adsorption of biological components (e.g. proteins) with a tremendous impact on the cellular response. The evaluation of these NPs is highly challenging and has produced some controversial results. Future research work should focus on the standardization of analytical or computational methodologies, aiming the identification of toxicity trends and the generation of a useful meta-analysis database on polymeric nanocarriers.This chapter covers all the aforementioned aspects, emphasizing the importance of the in vitro cellular studies in the first stages of polymeric nanocarriers development.