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© 2021 The Authors. Published by American Chemical Society, The frequency of brain disease has increased significantly in the past years. After diagnosis, therapeutic options are usually limited, which demands the development of innovative therapeutic strategies. The use of antibody−drug conjugates (ADCs) is promising but highly limited by the existence of the blood−brain barrier (BBB). To overcome the impermeability of this barrier, antibody fragments can be engineered and conjugated to BBB peptide shuttles (BBBpS), which are capable of brain penetration. Herein, we linked the highly efficient BBBpS, PepH3, to the IgG fragment crystallizable (Fc) domain using the streamlined expressed protein ligation (SEPL) method. With this strategy, we obtained an Fc-PepH3 scaffold that can carry different payloads. Fc-PepH3 was shown to be nontoxic, capable of crossing an in vitro cellular BBB model, and able to bind to the neonatal Fc receptor (FcRn), which is responsible for antibody long half-life (t1/2). Overall, we demonstrated the potential of Fc-PepH3 as a versatile platform readily adaptable to diverse drugs of therapeutic value to treat different brain conditions., We acknowledge the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP (Grant Nos. PD/BD/128281/2017, PTDC/BIA-BQM/5027/2020, and DL 57/2016/CP1451/CT0023), and “la Caixa” Banking Foundation (ID 100010434), under the agreement LCF/PR/HR17/5215001.

The biomedical application of discrete supramolecular metal-based structures, specifically self-assembled metallacages, is still an emergent field of study. Capitalizing on the knowledge gained in recent years on the development of 3-dimensional (3D) metallacages as novel drug delivery systems and theranostic agents, we explore here the possibility to target [Pd2L4]4+ cages (L = 3,5-bis(3-ethynylpyridine)phenyl ligand) to the brain. In detail, a new water-soluble homoleptic cage (CPepH3) tethered to a blood brain barrier (BBB)-translocating peptide was synthesized by a combination of solid-phase peptide synthesis (SPPS) and self-assembly procedures. The cage translocation efficacy was assessed by inductively coupled mass spectrometry (ICP-MS) in a BBB cellular model in vitro. Biodistribution studies of the radiolabeled cage [[99mTcO4]- ⊂ CPepH3] in the CD1 mice model demonstrate its brain penetration properties in vivo. Further DFT studies were conducted to model the structure of the [[99mTcO4]- ⊂ cage] complex. Moreover, the encapsulation capabilities and stability of the cage were investigated using the [ReO4]- anion, the "cold" analogue of [99mTcO4]-, by 1H NMR spectroscopy. Overall, our study constitutes another proof-of-concept of the unique potential of supramolecular coordination complexes for modifying the physiochemical and biodistribution properties of diagnostic species.

© 2022 American Society for Microbiology. All Rights Reserved., Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation in HIV-1-infected (HIV) patients. Furthermore, detection of the HIV-1 matrix protein p17 (p17) in the central nervous system (CNS) and its ability to form toxic assemblies in the brain have been recently confirmed. Here, we show for the first time, using both an in vitro blood-brain barrier (BBB) model and in vivo biodistribution studies in healthy mice, that p17 can cross the BBB. There is rapid brain uptake with 0.35%  0.19% of injected activity per gram of tissue (IA/g) 2 min after administration, followed by brain accumulation with 0.28%  0.09% IA/g after 1 h. The interaction of p17 with chemokine receptor 2 (CXCR2) at the surface of brain endothelial cells triggers transcytosis. The present study supports the hypothesis of a direct role of free p17 in neuronal dysfunction in HAND by demonstrating its intrinsic ability to reach the CNS., This study was supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC) grant 20108 (to A.C.) and by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement 828774 (to M.C.)

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery., This research was funded by: the Portuguese Funding Agency, Fundacão para a Ciência e a Tecnologia, FCT IP: IF/01010/2013 and PTDC/BBB-BIO/0508/2014 to FAS, SFRH/BPD/100522/2014 and DL57/2016/CP1438/CT0002 to SIA, PD/BD/128281/2017, PTDC/BIA-BQM/5027/2020 and Dl 57/2016/CP1451/CT0023 to MC and VN, UIDB/04138/2020 and UIDP/04138/2020 to MMG, UID/Multi/04349/2020 and PTDC/QUI-NUC/30147/2017 to LG and JDGC, UID/NEU/04539/2019, UIDB/04539/2020 and ViraVector (CENTRO-01-0145-FEDER-022095) to RJN and LPA. Gilead Génese 2019 has provided support through project PGG/050/2019 to JNRD. CIISA has provided support through Project UIDB/00276/2020, funded by FCT. FAS and JG dedicate this study to the memory of Professor Carlos F. Barbas 3rd, an outstanding mentor and friend.

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., Brain metastases (BM) are a frequent complication in patients with advanced stages of cancer, associated with impairment of the neurological function, quality of life, prognosis, and survival. BM treatment consists of a combination of the available cancer therapies, such as surgery, radiotherapy, chemotherapy, immunotherapy and targeted therapies. Even so, cancer patients with BM are still linked to poor prognosis, with overall survival being reported as 12 months or less. Intercellular communication has a pivotal role in the development of metastases, therefore, it has been extensively studied not only to better understand the metastization process, but also to further develop new therapeutic strategies. Exosomes have emerged as key players in intercellular communication being potential therapeutic targets, drug delivery systems (DDS) or biomarkers. In this Review, we focus on the role of these extracellular vesicles (EVs) in BM formation and their promising application in the development of new BM therapeutic strategies., This research was funded by the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP, grants PD/BD/135046/2017, PTDC/BIA-BQM/5027/2020, DL 57/2016/CP1451/CT0023.

Friedel-Crafts acylation reaction was studied under mild conditions using hierarchical HY zeolite samples prepared through surfactant mediated technology, in the presence of NH4OH, using CTAB or DTAB as surfactants and changing the duration of treatment from 6 to 48 h. The materials were characterized by powder X-ray diffraction, low temperature N2 adsorption isotherms, SEM microscopy and pyridine adsorption followed by FTIR. The catalytic behaviour was studied in the acylation of furan by acetic anhydride. The catalytic results reflect the role of the duration of the treatment as well as the surfactant molecule used. As the time of treatment increases, the enlargement of the pores leads to an increase of the rate constant and turnover frequency (TOF), except for sample modified during longer time, 48 h, due to the occurrence of secondary reactions that produce larger products or reaction intermediates that become trapped inside the pores. The role of the surfactant molecule is also relevant since the sample modified in the presence of the larger surfactant molecule, CTAB, a significant increase in product yield and rate constant is obtained when compared with the sample prepared in the same conditions using DTAB. However, in that case, by prolonging the treatment a substantial decrease in the same parameters occurs due to the occurrence of deactivation phenomena, pointing out that the optimized modification of porosity needs to be customized according to the needs of each catalytic system.

© 2021 American Chemical Society, The activation of cannabinoid CB1 receptors (CB1R) by Δ9-tetrahydrocannabinol (THC), the main component of Cannabis sativa, induces analgesia. CB1R activation, however, also causes cognitive impairment via the serotonin 5HT2A receptor (5HT2AR), a component of a CB1R–5HT2AR heteromer, posing a serious drawback for cannabinoid therapeutic use. We have shown that peptides reproducing CB1R transmembrane (TM) helices 5 and 6, fused to a cell-penetrating sequence (CPP), can alter the structure of the CB1R–5HT2AR heteromer and avert THC cognitive impairment while preserving analgesia. Here, we report the optimization of these prototypes into drug-like leads by (i) shortening the TM5, TM6, and CPP sequences, without losing the ability to disturb the CB1R–5HT2AR heteromer, and (ii) extensive sequence remodeling to achieve protease resistance and blood–brain barrier penetration. Our efforts have culminated in the identification of an ideal candidate for cannabis-based pain management, an orally active 16-residue peptide preserving THC-induced analgesia., Rhodes Pharmaceuticals (L.P., Coventry, R.I., USA; 2016−2017), Marie Skłodowska-Curie Research and Innovation Staff Exchange (call H2020-MSCA-RISE-2014, 2015−2019), the Spanish Ministry of Economy and Innovation with FEDER funds (SAF2017-87629-R, PID2019-109240RB-I00), and the Fundació Bancària La Caixa (CaixaImpulse 2018 call; 2018−2020).

The biomedical application of discrete supramolecular metal-based structures, specifically self-assembled metallacages, is still an emergent field of study. Capitalizing on the knowledge gained in recent years on the development of 3-dimensional (3D) metallacages as novel drug delivery systems and

© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made., The incidence of metastatic breast cancer (MBC) is increasing and the therapeutic arsenal available to fight it is insufficient. Brain metastases, in particular, represent a major challenge for chemotherapy as the impermeable nature of the blood–brain barrier (BBB) prevents most drugs from targeting cells in the brain. For their ability to transpose biological membranes and transport a broad spectrum of bioactive cargoes, cell-penetrating peptides (CPPs) have been hailed as ideal candidates to deliver drugs across biological barriers. A more ambitious approach is to have the CPP as a drug itself, capable of both killing cancer cells and interacting with the blood/brain interface, therefore blocking the onset of brain metastases. vCPP2319, a viral protein-derived CPP, has both properties as it: (a) is selective toward human breast cancer cells (MDA-MB-231) and increases cell stiffness compared to breast epithelial cells (MCF 10A) hindering the progression of metastases; and (b) adsorbs at the surface of human brain endothelial cells potentially counteracting metastatic cells from reaching the brain. Overall, the results reveal the selective anticancer activity of the peptide vCPP2319, which is also able to reside at the blood–brain interface, therefore counteracting brain penetration by metastatic cancer cells., FDO, MC, and DG acknowledge FCT I.P. for fellowships PD/BD/135046/2017, PD/BD/128281/2017, and SFRH/ BPD/73500/2010. FCT I.P. is also acknowledged for funding (Projects PTDC/BIA-BQM/5027/2020 and PTDC/BBB-NAN/1578/2014). Marie Skøodowska-Curie Research and Innovation Staff Exchange (RISE) is also acknowledged for funding: call H2020-MSCARISE-2014, grant agreement 644167, 2015–2019. Work at UPF was supported by grant AGL2017-84097-C2-2-R and the “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0370) from the Spanish Ministry of Economy and Competitiveness (MINECO).

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide–drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide–porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections., Work supported by the La Caixa Health Foundation (project HR17_00409, ID 100010434, agreement LCF/PR/HR17/ 52150011), by the European Union (H2020-FETOPEN-2018- 2019-2020-01 grant no 828774), and by the Spanish Ministry of Science and Innovation (AEI/FEDER grant CTQ2017- 84371-P). NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB. Additional funding from Fundaca̧ o para a Cie ̃ ncia e Tecnologia (FCT-MCTES) is ̂ also acknowledged for DA Mendonca (PD/BD/136752/2018) ̧ and for C. Cruz-Oliveira and I. Cadima-Couto (PTDC/BIAVIR/29495/2017).

Copyright © 2020 Cavaco, Pérez-Peinado, Valle, Silva, Correia, Andreu, Castanho and Neves. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms, The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood–brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides., The authors thank the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP, for financial support (grants: PD/BD/128281/2017, PTDC/BBB-NAN/1578/2014); European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 828774; and “la Caixa” Banking Foundation under the project code HR17-00409.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)., The incidence of brain metastases (BM) in cancer patients is increasing. After diagnosis, overall survival (OS) is poor, elicited by the lack of an effective treatment. Monoclonal antibody (mAb)-based therapy has achieved remarkable success in treating both hematologic and non-central-nervous system (CNS) tumors due to their inherent targeting specificity. However, the use of mAbs in the treatment of CNS tumors is restricted by the blood-brain barrier (BBB) that hinders the delivery of either small-molecules drugs (sMDs) or therapeutic proteins (TPs). To overcome this limitation, active research is focused on the development of strategies to deliver TPs and increase their concentration in the brain. Yet, their molecular weight and hydrophilic nature turn this task into a challenge. The use of BBB peptide shuttles is an elegant strategy. They explore either receptor-mediated transcytosis (RMT) or adsorptive-mediated transcytosis (AMT) to cross the BBB. The latter is preferable since it avoids enzymatic degradation, receptor saturation, and competition with natural receptor substrates, which reduces adverse events. Therefore, the combination of mAbs properties (e.g., selectivity and long half-life) with BBB peptide shuttles (e.g., BBB translocation and delivery into the brain) turns the therapeutic conjugate in a valid approach to safely overcome the BBB and efficiently eliminate metastatic brain cells., This research was funded by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP, grants PD/BD/128281/2017, PTDC/BBB-BQB/1693/2014 and PTDC/BBB-NAN/1578/2014.

© 2017 American Chemical Society, The delivery of therapeutic molecules to the central nervous system is hampered by poor delivery across the blood-brain barrier (BBB). Several strategies have been proposed to enhance transport into the brain, including invasive techniques and receptor-mediated transport (RMT). Both approaches have several drawbacks, such as BBB disruption, receptor saturation, and off-target effects, raising safety issues. Herein, we show that specific domains of Dengue virus type 2 capsid protein (DEN2C) can be used as trans-BBB peptide vectors. Their mechanism of translocation is receptor-independent and consistent with adsorptive-mediated transport (AMT). One peptide in particular, named PepH3, reaches equilibrium distribution concentrations across the BBB in less than 24 h in a cellular in vitro assay. Importantly, in vivo biodistribution data with radiolabeled peptide derivatives show high brain penetration. In addition, there is fast clearance from the brain and high levels of excretion, showing that PepH3 is a very good candidate to be used as a peptide shuttle taking cargo in and out of the brain., The authors thank the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP, for financial support (grants SFRH/BPD/94466/2013; SFRH/BPD/109010/2015; IF/01010/2013; PTDC/BBBNAN/1578/2014; HIVERA/ 0002/2013) and Marie Skłodowska-Curie Research and Innovation Staff Exchange (MSCA-RISE), call 20-MSCARISE-2014 (grant agreement H20 644167 − INPACT). M.M., L.G., C.F., and J.D.G.C. gratefully acknowledge FCT support through the UID/Multi/04349/2013 project.

International audience; Carbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT-RNA) is presented. Fixed cells, previously exposed to oxDWNT-RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT-RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT-RNA with the small GTP-binding protein (Rab 11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network.

We have designed a drug delivery system for the anti-cancer drugs doxorubicin and mitoxantrone based on carbon nanotubes, which is stable under biological conditions, allows for sustained release, and promotes selectivity through an active targeting scheme. Carbon nanotubes are particularly promising for this area of application due to their high surface area, allowing for high drug loading, and their unique interaction with cellular membranes. We have taken a systematic approach to PEG conjugation in order to create a formulation of stable and therapeutically effective CNTs. The presented drug delivery system may be a means of improving cancer treatment modalities by reducing drug-related side effects.

Single-walled carbon nanotubes (SWCNTs) have been identified as a transporter for anti-cancer drugs, as they are capable of penetrating mammalian cell membranes and allow for a high drug loading due to their nanoscale dimensions and high aspect ratio. In addition, they can assist the targeting of therapeutic agents to the desired site of action by conjugation to antibodies or ligands of cancer cell surface receptors, which increases the effectiveness of the treatment and reduces side effects. In this work, we present a method for the triple functionalisation of oxidised SWCNTs with the anti-cancer drug doxorubicin, a monoclonal antibody, and a fluorescent marker at non-competing binding sites. The proposed methodology allows for the targeted delivery of the anti-cancer drug to cancer cells and the visualisation of the cellular uptake of SWCNTs by confocal microscopy. We show that the complex is efficiently taken up by cancer cells with subsequent intracellular release of doxorubicin, which then translocates to the nucleus while the nanotubes remain in the cytoplasm.

Mucosal immunization is advantageous over other routes of antigen delivery because it can induce both mucosal and systemic immune responses. Our goal was to develop a mucosal delivery vehicle based on bacteria generally regarded as safe, such asLactobacillusspp. In this study, we used the Lyme disease mouse model as a proof of concept. We demonstrate that an oral vaccine based on live recombinantLactobacillus plantarumprotects mice from tick-transmittedBorrelia burgdorferiinfection. Our method of expressing vaccine antigens inL. plantaruminduces both systemic and mucosal immunity after oral administration. This platform technology can be applied to design oral vaccine delivery vehicles against several microbial pathogens.

Este trabalho procura explorar as relações entre os hábitos de sono, os níveis de sonolência diurna e as queixas de ansiedade, depressão e stresse em adolescentes portugueses. 1613 jovens entre os 12 e os 18 anos (média=14,23, DP=1,752) responderam ao Questionário para Adolescentes AS, que incluía variáveis demográficas e sobre hábitos de sono, queixas de saúde, uma escala de ansiedade, depressão e stresse (EADS 21) e uma escala de sonolência (CASQ). 53,3% dos participantes eram do sexo feminino. Os resultados indicam que os níveis de sonolência diurna e os sintomas de depressão (Rs=0,371), ansiedade (Rs=0,327) e sobretudo stresse (Rs=0,405) estão significativamente correlacionados. Os jovens que apresentam maior sonolência diurna e sintomas de ansiedade, depressão e stresse são os que têm piores hábitos de higiene do sono, mais dificuldades em dormir bem ou adormecer, excesso de peso ou dores de cabeça. Conclui-se que a promoção de hábitos de sono saudáveis nos jovens será um factor importante na prevenção/tratamento de sintomas de depressão, ansiedade e stresse.

Carbon nanotubes (CNTs) exhibit unique size, shape and physical properties, which make them promising candidates for biomedical applications. In particular, carbon nanotubes have been intensively studied for conjugation with pre-existing therapeutic agents for more effective targeting, as a result of their ability to cross cell membranes. However, to utilise them effectively in biological systems it is extremely important to understand how they behave at the cellular level and their distribution in vivo. Additionally, in order to consider carbon nanotubes as candidate delivery systems of therapeutic agents it is important to ascertain their fate in vivo, but also take into account many factors, such as solubility, stability and clearance. Issues surrounding their short term and long term safety are currently the subject of toxicology testing. Herein, we propose to summarize the main findings on the uptake, trafficking and biodistribution of carbon nanotubes, with special focus on functionalized carbon nanotubes for delivery of therapeutic agents.

Approximately every fourth person in the world currently dies of cancer. Although many efficient anticancer drugs have been developed over the last 60 years or more, most therapeutic approaches still lack specificity for their intended site of action in the body, resulting in reduced effectiveness and severe side effects. The emerging field of nanomedicine provides a whole range of materials and techniques to develop customizable drug delivery vehicles that assist the targeting of therapeutic agents to the desired site of action. Amongst these, carbon nanotubes have emerged as promising candidates, being capable of penetrating mammalian cell membranes and allowing for the attachment of high loads of drugs and targeting agents on their surface or the inner cavity. This chapter will discuss the principles of targeted, anticancer chemotherapies and introduce carbon nanotubes as novel tools for vector-based, targeted drug delivery.

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Efforts to develop carbon nanotubes (CNTs) as nano-vehicles for precise and controlled drug and gene delivery, as well as markers for in vivo biomedical imaging, are currently hampered by uncertainties with regard to their cellular uptake, their fate in the body, and their safety. All of these processes are likely to be affected by the purity of CNT preparation, as well as the size and concentration of CNTs used, parameters that are often poorly controlled in biological experiments. It is demonstrated herein that under the experimental conditions of standard transfection methods, DWNTs are taken up by cultured cells but are then released after 24 h with no discernable stress response. The results support the potential therapeutic use of CNTs in many biomedical settings, such as cancer therapy.

Consumed for centuries, lactic acid bacteria are excellent candidates for the development of safe mucosal delivery vehicles for prophylactic and therapeutic molecules. We have recently reported that the immune response to an effective OspA-expressing L. plantarum vaccine for Lyme disease is modulated by the lipid modification of the antigen. In this study, we investigated if this technology can be applied to developing vaccines for other diseases by focusing on the Class A select agent, Yersinia pestis . We used a number of biochemistry and immunology techniques to determine the localization of the immunogen in our delivery vehicle and to evaluate the mucosal as well as the systemic immune response to the immunogen. We found that only LcrV cloned downstream of the signal sequence of B. burgdorferi OspA ( ss LcrV), but not wildtype LcrV (LcrV), is localized to the desired peptidoglycan layer of the delivery vehicle. In addition, only mice that received L. plantarum expressing ss LcrV produced significant titers of IgG antibody as well as IgA in distant mucosal sites such as lungs and vagina. Furthermore, only L. plantarum expressing ss LcrV induced significant amounts of pro-inflammatory cytokines TNFα, IL-12, IFNγ and IL-6 as well as anti-inflammatory IL-10 in human peripheral blood mononuclear cells derived dendritic cells, suggesting that the mechanism by which LcrV-expressing L. plantarum stimulates the immune response involves polarization to Th1 mediated immunity with some involvement of Th2. The study reported here proves that this system is a platform technology to develop oral vaccines for multiple diseases.

Aqueous dispersions of functionalized carbon nanotubes (CNTs) are now widely used for biomedical applications. Their stability in different in vitro or in vivo environments, however, depends on a wide range of parameters, such as pH and salt concentrations of the surrounding medium, and length, aspect ratio, surface charge, and functionalization of the applied CNTs. Although many of these aspects have been investigated separately, no study is available in the literature to date, which examines these parameters simultaneously. Therefore, we have chosen five types of carbon nanotubes, varying in their dimensions and surface properties, for a multidimensional analysis of dispersion stability in salt solutions of differing pH and concentrations. Furthermore, we examine the dispersion stability of oxidized CNTs in biological fluids, such as cellular growth media and human plasma, and their toxicity toward cancer cells. To enhance dispersibility and biocompatibility, the influence of different functionalization schemes is studied. The results of our investigations indicate that both CNT dimensions and surface functionalization have a significant influence on their dispersion and in vitro behavior. In particular, factors such as a short aspect ratio, presence of oxidation debris and serum proteins, low salt concentration, and an appropriate pH are shown to improve the dispersion stability. Furthermore, covalent surface functionalization with amine-terminated polyethylene glycol (PEG) is demonstrated to stabilize CNT dispersions in various media and to reduce deleterious effects on cultured cells. These findings provide crucial data for the development of biofunctionalization protocols, for example, for future cancer theranostics, and optimizing the stability of functionalized CNTs in varied biological environments.

Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

Early diagnosis of Lyme disease (LD) is critical to successful treatment. However, current serodiagnostic tests do not reliably detect antibodies during early infection. OspC induces a potent early immune response and is also one of the most diverse proteins in the Borrelia proteome. Yet, at least 70% of the amino acid sequence is conserved among all 21 known OspC types. We performed a series of comprehensive seroprofiling studies to select the OspC types that have the most cross-reactive immunodominant epitopes. We found that proteins belonging to seven OspC types detect antibodies from all three infected host species regardless of the OspC genotype of the infecting strain. Although no one OspC type identifies all seropositive human samples, combinations of as few as two OspC proteins identified all patients that had anti-OspC antibodies.

A doença falciforme é a enfermidade genética mais prevalente em todo o mundo. No Brasil, ela ocorre em um a cada 1.200 nascimentos. Por essa alta prevalência, ela representa, em nosso país, um importante problema de saúde pública. Além da hemólise, a vasoclusão é o achado central da doença e responsável pelas crises dolorosas, que correspondem à principal causa de internação nos adultos. Alguns pacientes evoluem com seis ou mais episódios dolorosos por ano. As crises graves e persistentes constituem, nesses pacientes, fator de mau prognóstico e um fator preditivo para morte precoce. Este artigo enfoca as abordagens terapêuticas das crises vasoclusivas nos pacientes de doença falciforme. Sickle cell disease (SCD) is the most prevalent genetic disease in the world. In Brazil it occurs in one in every 1200 births. This high prevalence makes SCD a very important public health problem in Brazil. Vaso-occlusion and hemolysis are the hallmarks of the disease. Vaso-occlusion results in painful episodes which are the main cause of hospitalization among adults with SCD. Some patients experience episodes as often as 6 times per year. Persistent, severe sickle cell pain is a poor prognostic sign and a predictor for early death. The management of vaso-occlusion episodes is discussed here, as well as the related complications.

International audience; Double-walled carbon nanotubes (DWNTs) prepared by catalytic chemical vapour deposition were functionalized in such a way that they were optimally designed as a nano-vector for the delivery of small interfering RNA (siRNA), which is of great interest for biomedical research and drug development. DWNTs were initially oxidized and coated with a polypeptide (Poly(Lys:Phe)), which was then conjugated to thiol-modified siRNA using a heterobifunctional cross-linker. The obtained oxDWNT-siRNA was characterized by Raman spectroscopy inside and outside a biological environment (mammalian cells). Uptake of the custom designed nanotubes was not associated with detectable biochemical perturbations in cultured cells, but transfection of cells with DWNTs loaded with siRNA targeting the green fluorescent protein (GFP) gene, serving as a model system, as well as with therapeutic siRNA targeting the survivin gene, led to a significant gene silencing effect, and in the latter case a resulting apoptotic effect in cancer cells.