Suchergebnisse

Rheumatoid arthritis is a common chronic inflammatory and destructive arthropathy that consumes considerable personal, social and economic costs. It consists of a syndrome of pain, stiffness and symmetrical inflammation of the synovial membrane (synovitis) of freely moveable joints such as the knee (diarthrodial joints). Although the etiology of rheumatoid arthritis is unclear, the disease is characterized by inflammation of the synovial lining of diarthrodial joints, high synovial proliferation and an influx of inflammatory cells, macrophages and lymphocytes through angiogenic blood vessels. Disease-modifying antirheumatic drugs slow disease progression and can induce disease remission in some patients. Methotrexate is the first line therapy, but if patients become intolerant to this drug, biologic agents should be used. The development of biological substances for the treatment of rheumatic conditions has been accompanied by ongoing health economic discussions regarding the implementation of these highly effective, but accordingly, highly priced drugs are the standard treatment guidelines of rheumatic diseases. In this way, more efficient strategies have to be identified. Despite numerous reviews in rheumatoid arthritis in the last years, this area is in constant development and updates are an urgent need to incorporate new advances in rheumatoid arthritis research. This review highlights the immunopathogenesis rationale for the current therapeutic strategies in rheumatoid arthritis. ; Eugenia Nogueira (SFRH/BD/81269/2011) holds a scholarship from Fundacao para a Ciencia e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the ...

Liposomes have gained extensive attention as carriers for a wide range of drugs due to being both nontoxic and biodegradable as they are composed of substances naturally occurring in biological membranes. Active targeting for cells has explored specific modification of the liposome surface by functionalizing it with specific targeting ligands in order to increase accumulation and intracellular uptake into target cells. None of the Food and Drug Administration-licensed liposomes or lipid nanoparticles are coated with ligands or target moieties to delivery for homing drugs to target tissues, cells or subcellular organelles. Targeted therapies (with or without controlled drug release) are an emerging and relevant research area. Despite of the numerous liposomes reviews published in the last decades, this area is in constant development. Updates urgently needed to integrate new advances in targeted liposomes research. This review highlights the evolution of liposomes from passive to active targeting and challenges in the development of targeted liposomes for specific therapies. ; Eugenia Nogueira (SFRH/BD/81269/2011) holds a scholarship from Fundacao para a Ciencia e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This study was also supported by FEDER through POFC-COMPETE and by national funds from FCT through the project PEst-C/BIA/UI4050/2014 and the strategic funding of ID/BIO/04469/2013 unit. This work was supported by FCT I.P. through the strategic funding ...

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease, affecting almost 1% of the world population. Although the cause of RA remains unknown, the complex interaction between immune mediators (cytokines and effector cells) is responsible for the joint damage that begins at the synovial membrane. Activated macrophages are critical in the pathogenesis of RA and have been shown to specifically express a receptor for the vitamin folic acid (FA), folate receptor (FR). This particular receptor allows internalization of FA-coupled cargo. In this review we will address the potential of nanoparticles as an effective drug delivery system for therapies that will directly target activated macrophages. Special attention will be given to stealth degree of the nanoparticles as a strategy to avoid clearance by macrophages of the mononuclear phagocytic system (MPS). This review summarizes the application of FA-target nanoparticles as drug delivery systems for RA and proposes prospective future directions. ; Eugénia Nogueira (SFRH/BD/81269/2011) holds a scholarship from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project "BioHealth — Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER. This work was also supported by FCT I.P. through the strategic funding UID/BIA/04050/2013. We thank the Centro Hospitalar do Alto Ave (Guimarães, Portugal) for providing radiographic joint ...

Although the anti-cancer properties of 3BP have been described previously, its selectivity for cancer cells still needs to be explained. In the work reported here we characterized the kinetic parameters of radiolabelled [14C]-3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0 the affinity of cancer cells for 3BP transport, correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the proton motive force and is decreased by MCTs inhibitors. In the SK-BR-3 cell line, a sodium-dependent transport also occurs. Butyrate promotes the localization of MCT-1 at the plasma membrane and increases the level of MCT-4 expression, leading to a higher sensitivity for 3BP. Here, we demonstrate that this phenotype is accompanied by an increase in affinity for 3BP uptake. Our results confirm the role of MCTs, especially MCT-1 in 3BP uptake and the importance of CD147 glycosylation in this process. We find that the affinity for 3BP transport is higher when the extracellular milieu is acid. This is a typical phenotype of tumor microenvironment and explains the lack of secondary effects of 3BP already described in in vivo studies. ; FEDER (Fundo Europeu deDesenvolvimento Regional), through POFC (Programa Operacional Factores de Competitividade) – COMPETE, and by Portuguese National Funds from FCT (Fundac¸˜ao para a Ciˆencia e Tecnologia) in the scope of the project PEst-OE/BIA/U14050/2014. JAS [grant number SFRH/BD/76038/2011] received a fellowship from the Portuguese government from the FCT through FSE (Fundo Social Europeu) and POPH (Programa Operacional Potencial ...

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, are reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly. ; SFRH/BD/81479/2011 and SFRH/BD/81269/2011 scholarships from Fundação para a Ciência e a Tecnologia (FCT). This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project ...

Cationic liposomes are efficient vectors for systemic delivery of therapeutic small interfering RNA (siRNA), taking advantage of RNA interference (RNAi), a naturally occurring gene-silencing mechanism in mammalian cells. However, toxicity at high concentrations, short circulating half-lives and lack of specificity restrict their successful application in a wider scale. The purpose of this study was to evaluate the efficiency of neutral liposomes containing polyethylene glycol (PEG) to encapsulate siRNA in their aqueous core. This formulation will reduce drastically the toxicity associated to cationic liposomes by bringing surface charge to almost zero, increasing stealth degree and therefore circulation time. In this study, we evaluate the efficiency of folate-targeted liposomes for specific delivery of siRNA to activated macrophages, key effector cells in rheumatoid arthritis (RA) pathology which specifically express folate receptor (FR). Myeloid cell leukaemia-1 (Mcl-1) is a protein essential for synovial macrophage survival, since Mcl-1 suppression results in the induction of apoptosis. The effect of MCL1 siRNA incorporated in liposomal formulation was assessed in primary human macrophages and successful inhibition of Mcl-1 expression was achieved. Here we show that the neutral liposomal derived from DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) formulation developed is efficient to encapsulate MCL1 siRNA and silencing gene expression in activated human macrophages. ; Eugénia Nogueira (SFRH/BD/81269/2011) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project "BioHealth − Biotechnology and Bioengineering approaches to improve health quality", Ref. ...

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34,580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells. ; Eugénia Nogueira (SFRH/BD/81269/2011), Célia F. Cruz (SFRH/BD/ 100927/2014) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This study was also supported by FEDER through POFC – COMPETE and by national funds from FCT through the project PEst-UID/ BIA/4050/2013 and the strategic funding of ID/BIO/04469/2013 unit.We thank the Immuno-haemotherapy Department of Hospital de São João (Porto, Portugal) for providing buffy coats from healthy ...

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers. ; This work has received funding from the European Union's Seventh Framework Program (FP7/2007-2013; grant agreement NMP4-LA-2009-228827 NANOFOL) and Horizon 2020 Research and Innovation Program (grant agreement No 683356 - FOLSMART), further from the Portuguese Foundation for Science and Technology under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020. ...

Bovine Serum Albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5 mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100 nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific Folate Receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy. ; European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC – COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014. The authors also thank the FCT Strategic Project of UID/BIO/04469/2013 ...

Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biological therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor present at the surface of activated macrophages, key effector cells in this pathology. The specificity of our liposomal formulation to target folate receptor was investigated both in vitro as in vivo using a mouse model of arthritis (collagen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor . These liposomal formulations also significantly increase the clinical benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance. ; Eugenia Nogueira (SFRH/BD/81269/2011), Ana Loureiro (SFRH/BD/81479/2011) and Catarina Almeida (SFRH/BPD/48533/2008) hold scholarships from Fundacao para a Ciencia e a Tecnologia (FCT). Goncalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This study was also supported by FEDER through POFC-COMPETE, by national funds from FCT through the ...

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol. ; Eugenia Nogueira (SFRH/BD/81269/2011) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundacao para a Ciencia e a Tecnologia (FCT). Goncalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. This work was also supported by FCT I.P. through the strategic ...