The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.

Particle engineering for co-delivery of drugs has the potential to combine multiple drugs with different pharmaceutical mechanisms within the same carrier, increasing the therapeutic efficiency while improving patient compliance. This work proposes a novel approach for producing polymer–polymer core–shell microparticles by multi-step processing of emulsion and spray drying. The particle core was obtained by an oil-in-water emulsion of poly(ε-caprolactone) (PCL) loaded with curcumin (CM), followed by the resuspension in poly(vinyl alcohol) (PVA) containing ciprofloxacin (CPx) forming the shell layer by spray-drying. The obtained core-shell particles showed an average size of 3.8 ± 1.2 μm, which is a suitable size for inhalation therapies. The spatial distribution of the drugs was studied using synchrotron-based macro attenuated total reflection Fourier transform infrared (macro ATR-FTIR) microspectroscopy to map the chemical distribution of the components within the particles and supported the presence of CM and CPx in the core and shell layers, respectively. The formation of the core–shell structure was further supported by the differences in the release profile of CM from these particles, when compared to the release profile observed for the single particle structure (PCL-CM). Both empty and drug-loaded carriers (up to 100 μg.mL−1) showed no cytotoxic effects on A549 cells while exhibiting the antibacterial activity of CPx against Gram-positive and Gram-negative bacteria. These polymer core–shell microparticles provide a promising route for the combination and sequential drug release therapies, with the potential to be used in inhalation therapies

O cancro colorretal (CCR), caracterizado por afetar o cólon ou o reto, é marcado pela proliferação descontrolada de células anormais no revestimento destes órgãos. As modalidades de tratamento primárias contra o CCR englobam a intervenção cirúrgica e a quimioterapia, frequentemente não conseguem proporcionar resultados eficazes. Assim, existe uma necessidade urgente de terapias inovadoras e direcionadas, para melhorar o prognóstico e as taxas de sobrevivência dos doentes. A lactoferrina de origem bovina (bLf) é uma proteína natural derivada do leite que demonstrou uma toxicidade seletiva contra as células do CCR. No entanto, a ingestão oral de bLf pode levar à sua degradação, reduzindo a quantidade de bLf que consegue alcançar as células-alvo, diminuindo consequentemente o seu efeito terapêutico. Para ultrapassar estas limitações, os exossomas, subtipo de vesículas extracelulares (VEs), representam uma potencial solução devido à sua versatilidade numa ampla gama de agentes terapêuticos. Nesta dissertação, procurou-se desenvolver uma estratégia para promover o potencial anticancerígeno da bLf, prevenindo a sua degradação gástrica e, assim, aumentando a sua biodisponibilidade e libertação controlada. A citotoxicidade da bLf livre foi testada em várias linhas celulares de CCR, nomeadamente RKO e HCT-116. Os exossomas foram caracterizados no que respeita à distribuição de tamanho, potencial zeta, concentração de partículas e presença dos marcadores de superfície. A incorporação de bLf nos exossomas foi efetuada por incubação direta e eletroporação, sendo esta última a que produziu maiores eficiências de encapsulação. A cromatografia de exclusão por tamanho foi usada para remover a bLf não encapsulada. A eficiência de encapsulação da bLf foi de 7,6 ± 0,4%, determinada através de UHPLC. O complexo Exos-bLf mostrou-se mais eficiente na redução da viabilidade celular de RKO e HCT-116 do que a bLf livre, apresentando também citotoxicidade mais rápida, após 24 h de tratamento. O sistema Exos-bLf foi submetido à digestão in vitro, o que permitiu confirmar que os exossomas protegeram a bLf da degradação, potenciando desta forma o seu efeito terapêutico. Após a digestão, a bLf livre apresentou uma bioacessibilidade de 69,1 ± 4,9% e uma estabilidade de 14,4 ± 1,8%. A amostra Exos-bLf, após a digestão, exibiu uma bioacessibilidade inferior a 56,8 ± 10,3%, mas uma estabilidade melhorada de 28,4 ± 2,1%. No geral, estes resultados sugerem que a combinação de bLf com exossomas é promissora para uma entrega direcionada de fármacos na terapia do CCR, permitindo uma maior eficácia e proteção da proteína contra a sua degradação.

Active packaging is an increasingly reliable technology for assuring the safety and maintenance/improvement of the organoleptic traits of the enclosed food products. Bio-based materials with a wide set of highly impressive characteristics were assembled through a simple and effective methodology to produce affordable edible antibacterial active films. Bacterial cellulose (BC) was used as film scaffold since it is an extremely pure polysaccharide that possesses noteworthy properties for food casing such as high toughness, shape retention and works as a stabilizing agent. Functionalization of BC was achieved through absorption of lactoferrin (LF), a bilobar protein mainly extracted from milk that possess a numerous plethora of activities, such as broad spectrum antibacterial effect, immunoregulatory properties, and also promotes bifidobacteria and intestinal epithelium growth. These films were extensively characterized in terms of their physicochemical characteristics, bactericidal efficiency and cytotoxicity. The LF absorption and de-absorption profiles of the BC films were registered. Surface free energy, water vapour permeability, attenuated total reflection Fourier transform infrared spectroscopy, dynamic mechanical analysis (in water saturated conditions) and swelling profile, and other critical characterization techniques were employed. The antibacterial properties were assessed through the determination of the specific growth rate impact, “live and dead” fluorescence, scanning electron microscopy and colony forming units’ count; using two model microorganisms (Escherichia coli and Staphylococcus aureus). The contact killing properties were evaluated for standalone films and for a specific case study (fresh sausage). Finally, the films cytotoxicity was determined after digestion in a dynamic mimetic artificial gastrointestinal digestive system. All attained results lead us to conclude that the obtained active edible films display an effective and significant antimicrobial activity against both Gram – and + and revealed no cytotoxicity, prior and after the gastrointestinal digestion. Thus, these new films present a high potential to safely functionalize the encased foods, while providing prophylactic properties. Acknowledgements: The authors acknowledge the Portuguese Foundation for Science and Technology (FCT, Portugal) for the financial support provided by the research grants SFRH/BD/64901/2009, SFRH/BD/63578/2009, SFRH/BPD/64958/2009, SFRH/BPD/64958/2009 for Jorge Padrão, Sara Gonçalves, João P. Silva and Vitor Sencadas respectively, and also for FCT Strategic Project PEst-OE/EQB/LA0023/2013

Lactoferrin (Lf) is a versatile iron-binding protein abundant in milk and colostrum that exhibits several interesting biological activities, from which its antifungal and anticancer activities are particularly promising. Since the molecular basis underlying these activities are still not fully elucidated, herein we aimed at achieving a more comprehensive knowledge on this subject, envisioning an improved application of Lf against fungal infections and cancer. To this end, a multidisciplinary approach using yeast and cancer cells as cellular models was carried out. Lf has been shown to exert a powerful antifungal activity against several species of yeasts and filamentous fungi. Increasing research has been focused on understanding the mechanisms underlying this activity towards the development of novel Lf-based antifungal strategies. For instance, Lf was demonstrated to induce an apoptotic-like regulated cell death process in both Saccharomyces cerevisiae and Candida albicans. To get further insights on this process, we sought to investigate whether autophagy, a degradative process by which cells deliver cellular components to the vacuole, plays a role in Lf-induced yeast cell death. Using pharmacological and genetic approaches, we demonstrate that Lf antifungal activity depends on autophagy activation. Lf has also been demonstrated to display a strong anticancer activity against several cancer types, and some underlying mechanisms have been proposed, including cell cycle arrest, apoptosis induction and metastasis/invasion inhibition. Given that Lf has high affinity to iron and iron metabolism is critical for cancer cells, we explored for the first time the iron-cancer-Lf axis. For that purpose, we assessed the effect of Lf on the expression and localization of iron metabolism related proteins in highly metastatic breast cancer and non-cancer cells. Moreover, their expression, as well as Lf antiproliferative capacity, were evaluated upon iron chelation and supplementation. Our results demonstrate that Lf modulates the expression and localization of some iron metabolism-related proteins, induces severe changes in cytoskeleton organization, and that iron chelation and supplementation change the effect of Lf in a breast cancer cell line. Overall, though further research is needed, our results point to the combination of Lf with autophagy inducers as a promising antifungal strategy, as well as the perturbation of iron homeostasis as a Lf mechanism against cancer cells.

Lactoferrin (Lf) is an iron-binding globular protein that belongs to the transferrin family. This protein is usually encountered in various mammalian secretions with special higher concentration in milk and colostrum. In recent years, Lf has gained great interest due to its multifunctionality and cytotoxic activity against various cell types such as bacteria, fungi, cancer cells, and even viruses. One of the most promising Lf abilities is its powerful antifungal activity against different pathogenic fungi, whose molecular basis is not yet fully elucidated. Studies aiming to deeper understand the mechanisms underlying Lf antifungal capacity will therefore aid in potentiating this effect. Previous reports on Lf yeast killing activity were performed in buffer lacking nutrients. Since these conditions are likely to induce autophagy, we hypothesized that Lf cytotoxicity could be intrinsically associated with this process. Autophagy is a process that allows the transport and degradation of damaged cellular components in the lysosome/vacuole with the purpose of recycling them to be reused as building blocks for other metabolic processes. This thesis aims to elucidate the interplay between Lf and autophagy and to explore the combination of Lf with autophagy inducers as a novel strategy of antifungal treatment. The results showed that Lf cytotoxicity towards yeast cells is significantly enhanced under various autophagy inducing conditions, such as nutrient or nitrogen starvation media. Furthermore, we found that Lf antifungal activity is only impaired in strains lacking specific components of the autophagic machinery, suggesting that it depends on specific components/phases of autophagy. Our data also point to the conclusion that the combination of Lf with specific autophagy inducers, namely rapamycin and tunicamycin, increases Lf cytotoxicity indicating it may be a promising strategy for the treatment of fungal infections. Overall, these results further elucidate the mechanism of action behind Lf antifungal activity and its association with the autophagy process. In the future, it would be interesting to test the most promising combinations of Lf and autophagy inducers against human pathogenic yeast strains and clinical isolates to evaluate their efficiency as an antifungal strategy.

Lactoferrin has for long captured the interest of many researchers as a natural compound with a wide variety of uses. Lactoferrin is a monomeric, iron-binding 80 kDa glycoprotein, and appears to be the subfraction of whey with the best documented antiviral, antimicrobial, anticancer and immune modulating/enhancing effects. It belongs to the family of transferrin proteins, and serves to control iron levels in body fluids by sequestering and solubilizing ferric iron. In the present research effort, production of lactoferrin derivatives (starting from a purified commercial extract), encompassing full stabilization of its three-dimensional structure, has been attempted via nanoencapsulation within lipid nanovesicles, integrating a multiple water-in-oil-in-water emulsion. Long-term storage of the multiple nanoemulsions produced did not lead to leaching of protein, thus proving the effectiveness of the encapsulation procedure. Furthermore, lactoferrin nanovesicle derivatives prepared under optimal conditions were successfully employed at lab-scale antimicrobial trials.

The aim of this work was to evaluate the neutrophil activation state in chronic kidney disease (CKD) patients under hemodialysis, and its linkage with resistance to recombinant human erythropoietin (rhEPO) therapy. Methods: We studied 63 CKD patients under hemodialysis and rhEPO treatment (32 responders and 31 non-responders to rhEPO therapy). In 20 of the CKD patients (10 responders and 10 non-responders to rhEPO therapy), blood samples were also collected immediately after dialysis. Twenty-six healthy volunteers were included in a control group. Hemoglobin levels, total and differential leukocyte counts, and circulating levels of C-reactive protein (CRP), elastase and lactoferrin were measured in all patients and controls. Results: Compared with controls, CKD patients presented with significantly higher CRP, neutrophil and elastase levels. When we compared the 2 groups of patients, we found that non-responders presented statistically significantly higher elastase plasma levels. A positive significant correlation was found between elastase levels and weekly rhEPO dose and CRP serum levels. After the hemodialysis procedure, a statistically significant rise in elastase, lactoferrin and, elastase/neutrophil and lactoferrin/neutrophil ratios were found. Conclusions: Our data show that CKD patients under hemodialysis present higher elastase levels (particularly in non-responding patients), which could be related to the rise in neutrophils, and to be part of the enhanced inflammatory process found in these patients

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a survival rate of 3 to 5 years from the onset of symptoms. ALS treatment is compromised by the existence of the blood-brain barrier (BBB), which restricts the access of promising biopharmaceutics to the brain, including riluzole, a drug commonly used to treat ALS. To circumvent the BBB and improve the drug brain targeting, nanosystems such as lipid nanoparticles can be employed. In this work, the permeation of nanostructured lipid carriers (NLC) loaded with riluzole and functionalized with a specific ligand – lactoferrin – was assessed in an in vitro BBB model (hCMEC/D3 cell line). Moreover, the effect of the NLC on the production and secretion of the pro-inflammatory cytokine human interleukin 1 alpha (IL-1a) by the cells was also quantified. The permeability studies across the hCMEC/D3 cell monolayers showed that free riluzole penetrated the BBB more than the riluzole-loaded NLC, which was also consistent with the results from the ELISA kit, with the free drug eliciting a higher IL-1a production. Despite these findings, the developed nanocarriers possessed good biocompatibility and stability, and could, therefore, be considered suitable for brain applications.

This work reports on the impact of subtle change of protein charge on coacervation and subsequent liquid–liquid phase separation between two oppositely charged globular proteins. For this purpose, a comparative study was conducted on the coacervation of lactoferrin (LF) with the two β-lactoglobulin (β-LG) isoforms. Upon mixing LF with an excess of β-LG, microspheres were formed throughout coacervation under narrow pH range (5.4–6.0). At the optimal pH of coacervation, LF being the limiting partner under tested concentration ranges. The β-LG/LF molar ratio recovered in the formed coacervates varied from 4 to 8 depending on the total protein concentration. Remarkably, LF showed a selective coacervation with isoform A of β-LG as judged by a larger concentration domain for coacervation and a high yield of LF recovered once mixed with β-LG A i.e. 80% against a maximum of 42% with β-LG B. At thermodynamic level, the interaction of LF with both β-LG isoforms exhibited complex exothermic binding isotherms with both enthalpic and entropic contributions.