Folate-targeted nanoparticles for rheumatoid arthritis therapy

Designer Biology: From Proteins and Cells to Scaffolds & MaterialsRheumatoid 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 showed specifically express a receptor for the vitamin folic acid (FA), folate receptor (FR). This particular receptor allows internalization of FA-coupled cargo. In this work 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 work summarizes the application of FA-target nanoparticles as drug delivery systems for RA and proposes prospective future directions.info:eu-repo/semantics/publishedVersio

Liposomal formulations for specific drug delivery

NANOFOL NanoDiaRA Conference in the 7th Framework Programme: Nanoparticles for Early Diagnostics of Inflammatory Diseases | New approaches in the field of soft and hard nanoparticlesLiposomes have received considerable scrutiny as possible vehicles for drug delivery due to properties such as sustained release, increased drug stability, ability to overcome drug resistance and targeting of specific tissues. In this study, we have produced several liposomal formulations prepared by thin film hydration method. The different formulations were extensively characterized and preliminary tests were performed to evaluate their potential as specific drug delivery systems. The liposomes present very small values of size and polydispersity index, as well as lower cytotoxicity, which are compatible with intended in vivo applications. The incorporation of imaging and targeting agents in the liposomes was performed, what improved selectivity to the system, being the liposomes specifically internalized in target cells. The encapsulation of drugs was also performed and their effect analyzed in the target cells. In summary, the liposomal formulations obtained exhibit suitable characteristics to be used in vivo as specific drug delivery systems.info:eu-repo/semantics/publishedVersio

Folate-targeted nanoparticles for rheumatoid arthritis therapy

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 images

Effect of sucrose in freeze-dried liposomes encapsulating drugs

XII Spanish-Portuguese Conference on Controlled Drug DeliveryThe use of liposomes as drug delivery system is very promising due to their ability to encapsulate hydrophilic and hydrophobic drugs [1]. However, the long-term storage of liposomes reveals physical and chemical instabilities which limits the use in therapeutic applications [2]. The development of a dry powder formulation can be a solution to improve these problems. The production of a freeze-dried liposomes encapsulating drugs is considered a key challenge, since the drugs can leak out from the liposomes during the freeze-drying process, occurring drug leakage [3]. The stress caused by the main steps of the process may affect the structure of liposomes. Therefore, cryoprotectants can be used to prevent damage in the integrity of the liposome bilayer [2]. The aim of this study was to optimize a liposomal formulation that after freeze-drying continues to be stable and able to maintain drugs with few leakages. The protective effect of five sugars at different concentration was tested in terms of size distribution, morphology and concentration. Results showed that sucrose, in a concentration dependent manner, effectively prevents liposomal fusion or aggregation and protects the integrity of freeze-dried liposomes (Figure 1). This liposomal formulation encapsulating a hydrophobic drug Tamoxifen can be freeze-dried and stored without significant drug leakage. The biological activity of drug after freeze-drying was also evaluated. In sum, the results indicated that this optimized liposomal formulation can be a good approach to long-term storage.This study was supported by the Portuguese Foundation for Science and Technology (FCT) 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 – Programa Operacional Regional do Norte. This work has also received funding from the European Union Horizon 2020 research and innovation programme under grant agreement NMP-06-2015- 683356 FOLSMART.info:eu-repo/semantics/publishedVersio

Folate-target nanodevices to activated macrophages for rheumatoid arthritis

6th Iberian Meeting on Colloids and InterfaceMethotrexate 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. In a previous European project NANOFOL, we performed 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. We 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. A new project, called FOLSMART, will perform the preclinical development and the phase I clinical trials of this new liposomal formulation.info:eu-repo/semantics/publishedVersio

Exposure assessment based recommendations to improve nanosafety at nanoliposome production sites

The NANOFOL concept aims at creating nanodevices containing a drug for inflammatory disorder treatment. This paper provides recommendations for nanosafety based on a measurement campaign which aimed at identifying exposure risks with respect to two specific phases of the products lifecycle, that is, production of the device and its waste management. The nanoparticles presence both in air and in liquid phase was studied. While no emissions were detected during the production period, many recommendations have been made, particularly regarding the nanowaste treatment, based on nanosafety guidelines.Eugenia Nogueira (SFRH/BD/81269/2011) holds scholarships from Fundacao para a Ciencia e a Tecnologia (FCT). The authors thank the EC for funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement NMP4-LA-2009-228827 NANOFOL

Desenvolvimento de lipossomas direcionados com folato para o tratamento de artrite reumatoide

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).Rheumatoid arthritis is a common, chronic inflammatory and destructive arthropathy with considerable personal, social and economic implications. Although the etiology of rheumatoid arthritis remains controversial, the hallmark of the disease is characterized by symmetrical inflammation of the synovial membrane of freely moveable joints, massive synovial proliferation and influx of inflammatory cells. Methotrexate is the first line therapy, but in intolerant patients biologic agents should be used. The development and clinical use of highly effective biological substances for standard treatment guidelines of rheumatic conditions treatment is a health economic problem due of being exceedingly costly. Activated macrophages are critical in the pathogenesis of rheumatoid arthritis and specifically express folate receptor β, a receptor for the vitamin folic acid. This receptor allows internalization of coupled cargo to folic acid by folate receptor-mediated endocytosis. Liposomes have gained extensive attention as carriers for a wide range of therapeutic agents because of being both nontoxic and biodegradable. This work aims to exploit the potential of folate-targeted liposomal nanoparticles as effective drug delivery systems for therapies directly targeting activated macrophages, reducing long term effects in rheumatoid arthritis patients. Specific targeting of activated macrophages is an important research challenge because they are phagocytic cells capable of internalizing any foreign particle. A complete stealth degree is needed to avoid the clearance of nanoparticles by macrophages of the reticuloendothelial system. Chapter II reports the study of surface modification of liposomes with the glycolipid monosialoganglioside and the polymer polyethylene glycol to improve stealth and decrease their clearance. Liposomes prepared with the monosialoganglioside clearly reduced macrophage uptake, but not to the same extent as liposomes prepared with 10% polyethylene glycol which ensure a proper stealth degree of the nanoparticles. The preparation of folate receptor-targeted liposomes, is typically performed by covalently attaching folic acid to a phospholipid or cholesterol anchor with polyethylene glycol and subsequent incorporation in the bilayer. Although these folic acid conjugates were shown to successfully target folate receptor α, there are some concerns regarding their use, namely chemical stability and self-aggregation at the liposome surface. Chapter III describes an innovative strategy for targeted liposomal delivery that uses a hydrophobic fragment of pulmonary surfactant protein D conjugated to a linker and folic acid. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity. The novel liposomal constructs are highly stable, specific for folate receptor α – expressing cells, and promote a more efficient liposomal membrane disruption than classic systems. The capacity of targeted liposomes to encapsulate and specifically deliver either hydrophobic or hydrophilic drugs into targeted cells was also demonstrated. The combination of all these characteristics of the new liposomes led us to evaluate its the efficiency to treat rheumatoid arthritis, by targeting folate receptor β present at the surface of activated macrophages. Chapter IV reports the encapsulation of methotrexate in the new liposomal formulation, to enhance its tolerance and efficacy. The presence of 10% polyethylene glycol in the liposomes greatly improved the stealth degree, promoting the specificity of folic acid-mediated targeting. Liposomes strongly accumulated in the joints of arthritic mice, demonstrating their specificity in vivo. Furthermore, this liposomal formulation significantly increases the clinical benefit and complete prophylactic efficacy of methotrexate in an animal model. Chapter V reports a preliminary study on the use of the optimized liposome nanoparticles as siRNA carriers. Myeloid cell leukaemia-1 protein is essential for synovial macrophage survival. This study demonstrated that neutral liposomal formulation derived from DOPE phospholipid ensures that siRNAs molecules, are encapsulated inside the core of the liposomes with high polyethylene glycol content, reducing the toxicity associated to cationic liposomes. Chapter VI describe the establishments of a method to assess the disruption of nanoparticle drug delivery system in vitro using Hoechst 34580 a DNA binding dye, as a drug mimicker. This approach enables to correlate the intensity of a fluorescent dye with the nanoparticle disruption and consequent drug delivery in the cytoplasm of the cell. In summary, this work reports the use of liposomes prepared with a novel peptide conjugate for folic acid-mediated delivery, what creates new opportunities for the treatment of human diseases, namely rheumatoid arthritis and cancer.A artrite reumatoide é uma artropatia inflamatória crónica comum e destrutiva, com consideráveis implicações pessoais, sociais e económicas. Embora a etiologia da artrite reumatoide permaneça controversa, a doença é caracterizada por inflamação simétrica da membrana sinovial de articulações móveis, intensa proliferação sinovial e influxo de células inflamatórias. O metotrexato é a terapia de primeira linha mas, em doentes intolerantes, devem ser usados agentes biológicos. O desenvolvimento e uso clínico de agentes biológicos altamente efetivos nas diretrizes de tratamento padrão de doenças reumáticas é um problema económico de saúde por serem altamente dispendiosos. Deste modo, estratégias mais eficazes têm de ser identificadas. Os macrófagos ativados têm papel vital na patogénese da artrite reumatoide e tem sido demonstrado que expressam especificamente o recetor de folato β, o recetor para a vitamina ácido fólico. Este recetor permite a internalização de ácido fólico acoplado a carga por endocitose. Os lipossomas são altamente atrativos como transportadores de vários agentes terapêuticos por serem não tóxicos e biodegradáveis. Este trabalho visa explorar o potencial dos lipossomas com folato como um sistema de libertação de fármacos eficaz para terapias direcionadas diretamente para macrófagos ativados, reduzindo efeitos a longo prazo em pacientes com artrite reumatoide. O direcionamento específico de macrófagos ativados é um importante desafio na investigação, porque estas células fagocíticas são capazes de internalizar qualquer partícula estranha. É necessário um grau de invisibilidade total, para evitar a remoção das nanopartículas por macrófagos do sistema reticuloendotelial. O Capítulo II reporta o estudo da modificação da superfície de lipossomas com o glicolípido monossialogangliosídeo e o polímero polietileno glicol para aumentar o grau de invisibilidade e diminuir a sua remoção. Os lipossomas preparados com o monossialogangliosídeo apresentam uma reduzida remoção pelos macrófagos, contudo não com a mesma extensão que lipossomas preparados com 10% de polietileno glicol, o qual assegura um grau de invisibilidade adequado nestas nanopartículas. A preparação de lipossomas direcionados para recetores de folato envolve normalmente a ligação covalente do ácido fólico a uma âncora de fosfolípido ou colesterol com polietileno glicol e sua incorporação na bicamada. Embora estes conjugados de folato tenham mostrado direcionar o vetor para o recetor de folato α, existem algumas preocupações subjacentes ao seu uso, nomeadamente estabilidade química e auto-agregação na superfície do lipossoma. O Capítulo III descreve uma estratégia inovadora para o direcionamento lipossomal que utiliza um fragmento hidrofóbico de proteína surfactante pulmonar D conjugada a um ligante e ácido fólico. O conjugado peptídico insere-se profundamente na bicamada lipídica sem afetar a integridade lipossomal. Estas novas construções são altamente estáveis, específicas para células que expressam o recetor de folato α e promovem a rutura da membrana lipossomal de forma mais eficiente do que o sistema clássico. Além disso, foi também demonstrada a capacidade destes lipossomas para encapsular e libertar especificamente nas células-alvo tanto fármacos hidrofóbicos como hidrofílicos. A combinação de todas estas características dos novos lipossomas levou-nos a avaliar a sua eficácia para o tratamento de artrite reumatoide, por direcionamento do recetor de folato β, presente na superfície de macrófagos ativados. O Capítulo IV relata o encapsulamento do metotrexato na nova formulação lipossomal para melhorar a sua tolerância e eficácia. A presença de 10% de polietileno glicol nos lipossomas melhorou significativamente o grau de invisibilidade, promovendo a especificidade de direcionamento mediada pelo ácido fólico. Os lipossomas acumulam-se fortemente nas articulações de ratinhos com artrite, demonstrando a sua especificidade in vivo. Além disso, esta formulação lipossomal aumenta significativamente o benefício clínico e apresenta uma total eficácia profilática do metotrexato num modelo animal. O Capítulo V reporta um estudo preliminar sobre a utilização dos lipossomas como transportadores de siRNA. A proteína myeloid cell leukaemia-1 é essencial para a sobrevivência de macrófagos do sinóvio. Este estudo demonstra que é possível utilizar uma formulação lipossomal neutra derivada do fosfolípido DOPE assegurando que os siRNAs são encapsulados no interior central dos lipossomas com concentração elevada de polietileno glicol, reduzindo a toxicidade associada aos lipossomas catiónicos. O Capítulo VI descreve o estabelecimento de um método para avaliar a disrupção de lipossomas como sistemas de libertação de fármacos in vitro, utilizando o Hoechst 34580, um corante que se liga ao DNA, para simular o fármaco. Esta abordagem permite correlacionar a intensidade de um corante fluorescente com a rutura das nanopartículas e consequente libertação da droga no citoplasma da célula. Em resumo, este trabalho reporta a utilização de lipossomas preparados com o novo conjugado peptídico, para direcionamento mediada por ácido fólico, abrindo novas oportunidades para o tratamento de doenças humanas, como artrite reumatoide e cancro.Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/81269/2011 e UID/BIA/04050/2013

Quantification of drugs encapsulated in liposomes by 1H NMR

Liposomes are one of the most important and extensively studied drug delivery system due to their ability to encapsulate different kinds of drugs. Exploiting the advantages of 1H Nuclear Magnetic Resonance (NMR) spectrometry, we established a rapid and easy method for quantification of drugs encapsulated in liposomes. An internal standard, pyridine, was used for quantitative determination of drug concentration. Two different drugs were involved in this work, one hydrophilic, methotrexate disodium salt, and another hydrophobic, tamoxifen. The specificity and selectivity of the suggested method were evaluated by the absence of overlapping of at least one signal of each drug with pyridine in the NMR spectrum. The accuracy and precision of the method were assessed by adding a known amount of each drug to unloaded liposomes. Results obtained by quantitative NMR (qNMR) were validated and confirmed by comparing with two other traditional techniques, Ultraviolet - Visible (UV-Vis) spectrophotometry and High-Performance Liquid Chromatography (HPLC). It was found that the results were consistent with the ones obtained from our proposed qNMR method. Considering all the experiments conducted in this study, we deliberate that qNMR can be a suitable tool for the determination of drugs encapsulated in liposomes.This work has received funding from the European Union Horizon 2020 research andinnovation programme undergrantagreement NMP06-2015-683356 FOLSMART. This study was also supported by the Portuguese Foundation for Science and Technology (FCT) 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 - Programa Operacional Regional do Norte. Diana Guimarães (SFRH/BD/140321/ 2018) and Jennifer Noro (SFRH/BD/121673/2016) holds a scholarship from FCT. SFRH/BD/121673/2016info:eu-repo/semantics/publishedVersio