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- Biocompatibility and stability of polysaccharide polyelectrolyte complexes aimed at respiratory deliveryPublication . Rodrigues, Susana; Cardoso, Lurdes; Costa, Ana M. Rosa da; Grenha, AnaChitosan (CS) and chondroitin sulfate (CHS) are natural polymers with demonstrated applicability in drug delivery, while nanoparticles are one of the most explored carriers for transmucosal delivery of biopharmaceuticals. In this work we have prepared CS/CHS nanoparticles and associated for the first time the therapeutic protein insulin. Fluorescein isothiocyanate bovine serum albumin (FITC-BSA) was also used to enable comparison of behaviors regarding differences in molecular weight (5.7 kDa versus 67 kDa). Nanoparticles of approximately 200 nm and positive zeta potential around +20 mV were obtained. These parameters remained stable for up to 1 month at 4 C. Proteins were associated with efficiencies of more than 50%. The release of FITC-BSA in PBS pH 7.4 was more sustained (50% in 24 h) than that of insulin (85% in 24 h). The biocompatibility of nanoparticles was tested in Calu-3 and A549 cells by means of three different assays. The metabolic assay MTT, the determination of lactate dehydrogenase release, and the quantification of the inflammatory response generated by cell exposure to nanoparticles have indicated an absence of overt toxicity. Overall, the results suggest good indications on the application of CS/CHS nanoparticles in respiratory transmucosal protein delivery, but the set of assays should be widened to clarify obtained results.
- In vitro behaviour of konjac glucomannan microparticles aimed at pulmonary tuberculosis therapyPublication . Guerreiro, Filipa; Swedrowska, Magda; Rosa Da Costa, Ana; Grenha, Ana; Forbes, BenTuberculosis is one of the highest causes of death worldwide. Long periods of treatment are required and result in some cases in therapeutic incompliance, potentiating the development of multidrug-resistant tuberculosis. Thus, new approaches to treat the infection are required as an alternative to conventional orally administered treatment. This work proposes the development of an inhalable system, which is specifically targeted to alveolar macrophages, where the Mycobacterium tuberculosis is located. The targeting is mainly driven by the selected matrix material, konjac glucomannan (KGM), a natural polymer comprising mannan units that are expected to potentiate phagocytosis. Microparticles were loaded with two antitubercular drugs, isoniazid (INH) and rifabutin (RFB). KGM/INH/RFB microparticles were produced by spray-drying to produce particles with suitable characteristics to deliver INH and RFB to the alveolar region. The KGM/INH/RFB microparticles possessed an aerodynamic diameter of approximately 3 µm, meeting the requirement of a therapy targeted to alveolar macrophages. Moreover, KGM microparticles exhibited suitable geometric size (2.24 µm) and shape (spherical) to be phagocytosed to deliver drugs to infected macrophages. INH and RFB were associated with KGM microparticles with efficiencies of 91% and 74%, respectively. Similar in vitro release profiles were observed for both drugs in simulated lung fluid (SLF) replicating the lining fluid composition found in human alveoli.
- Nanoencapsulation of Gla-Rich Protein (GRP) as a novel approach to target inflammationPublication . Viegas, Carla; Araújo, Nuna; Carreira, Joana; Pontes, Jorge Filipe; Macedo, Anjos L.; Vinhas, Maurícia; Moreira, Ana S.; Faria, Tiago Q.; Grenha, Ana; de Matos, António A.; Schurgers, Leon; Vermeer, Cees; Simes, DinaChronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP’s therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG’s anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1β, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.
- Sulphated locust bean gum-coated lipid nanocapsules as potential lung delivery carriersPublication . Pontes, Jorge Filipe; Braz, L.; Guerreiro, Filipa; Rosa Da Costa, Ana; Almouazen, Eyad; Lollo, Giovanna; Grenha, AnaDrugs pertaining to Biopharmaceutics Classification System (BCS) classes II and IV have limitations in their delivery, including in the lung. Therefore, drug delivery carriers have been proposed to improve the therapeutic effectiveness of such drugs. This work proposes lipid nanocapsules (LNC) as a potential platform for lung drug delivery. Locust bean gum (LBG), which is a galactomannan, was used as polymeric shell, protecting the oily core of the nanocapsules and providing their surface with hydrophilic character. Due to the neutral character of LBG, in order to enable nanocapsule formation, a sulphate derivative (LBGS) was prepared, which was confirmed by Fourier-transformed infrared (FTIR) spectroscopy. The electrostatic interaction between the negatively charged sulphate groups of LBGS and the positively charged groups of the used cationic lipid (1,2-dioleoyloxy-3- trimethylammoniumpropanchloride, DOTAP), allowed the formation of monodisperse nanocapsules, with sizes around 200 nm and strongly negative zeta potentials, between -70 and -85 mV. Envisaging potential lung drug delivery, the LBGS-coated LNC were co-formulated with mannitol using spray-drying, producing microencapsulated nanocapsules. Feret’s diameter was determined to be 2.6 ± 1.8 µm and 3.1 ± 1.9 µm for Man (control) and Man/LNC microparticles, respectively. Further studies are underway in order to optimise both the nanoplatform and the dry powder formulation.
- Nanoencapsulation as a novel delivery approach for therapeutic applications of gla-rich protein (GRP)Publication . Araújo, Nuna; Viegas, Carla; Pontes, Jorge Filipe; Marreiros, Catarina; Raimundo, Pedro; Macedo, Anjos L.; Alves de Matos, António; Grenha, Ana; Vermeer, Cees; Simes, DinaGla rich protein (GRP) is a vitamin K dependent protein, shown to function as an inhibitor of pathological calcification and as an anti-inflammatory agent, with potential therapeutic use for age-related diseases such as osteoarthritis (OA) [1,2]. OA is a leading cause of disability and morbidity in the older population and constitutes a major world wide challenge for our health system. Presently, there are no drugs approved that can prevent, stop, or even restrain progression of OA. GRP has been shown to be able to lower inflammation and mineralisation processes in the articular tissue. Chitosan/tripolyphosphate (TPP) nanoparticles were selected for this study due to their biocompatibility, biodegradability and capacity to overcome the problem of low solubility of GRP in physiological conditions. This study aims to produce and characterise chitosan/TPP nanoparticles as GRP-delivery vehicles and test its anti-inflammatory potential in human macrophages.
- The era of nanomedicinePublication . Grenha, AnaJournal of Pharmacy and Bioallied Sciences is now in its 7th issue and in the way that has been paved so far, it is possible to find some works on nanomedicine. This term encompasses the applications of nanotechnology in the diagnosis and treatment of diseases and comprises an emerging field that is believed to have the potential to revolutionise individual health in the present century. The outstanding approach of nanomedicine is that it offers the delivery of potential drugs which were previously beyond the capacities of microscale technologies, due to specific biological barriers. Currently, several nanotherapeutics are approved or are undergoing clinical trials and application of nanotechnologies is expected to extend to many more commercial products in the near future.
- Dual antibiotherapy of tuberculosis mediated by inhalable locust bean gum microparticlesPublication . Rodrigues, Susana; Alves, Ana D.; Cavaco, Joana S.; Pontes, Jorge Filipe; Guerreiro, Filipa; Rosa Da Costa, Ana; Buttini, Francesca; Grenha, AnaDespite the existence of effective oral therapy, tuberculosis remains a deadly pathology, namely because of bacterial resistance and incompliance with treatments. Establishing alternative therapeutic approaches is urgently needed and inhalable therapy has a great potential in this regard. As pathogenic bacteria are hosted by alveolar macrophages, the co-localisation of antitubercular drugs and pathogens is thus potentiated by this strategy. This work proposes inhalable therapy of pulmonary tuberculosis mediated by a single locust bean gum (LBG) formulation of microparticles associating both isoniazid and rifabutin, complying with requisites of the World Health Organisation of combined therapy. Microparticles were produced by spray-drying, at LBG/INH/RFB mass ratio of 10/1/0.5. The aerodynamic characterisation of microparticles revealed emitted doses of more than 90% and fine particle fraction of 38%, thus indicating the adequacy of the system to reach the respiratory lung area, thus partially the alveolar region. Cytotoxicity results indicate moderate toxicity (cell viability around 60%), with a concentration-dependent effect. Additionally, rat alveolar macrophages evidenced preferential capture of LBG microparticles, possibly due to chemical composition comprising mannose and galactose units that are specifically recognised by macrophage surface receptors. (C) 2017 Elsevier B.V. All rights reserved.
- Pullulan-based nanoparticles: future therapeutic applications in transmucosal protein deliveryPublication . Rodrigues, Susana; Grenha, AnaCurrent advances in the field of drug delivery are aimed at finding adequate strategies for the administration of different drugs. Transmucosal delivery is the first-line option for the systemic delivery of many drugs, including proteins, a group of molecules with recognised therapeutic potential and wide range of applications (drug and genetic therapy, immunisation, among others).
- Chitosan and its derivatives as nanocarriers for siRNA deliveryPublication . Al-Qadi, Sonia; Grenha, Ana; Remuñán-López, CarmenThe ability to specifically silence genes using siRNA has enormous potential for treating genetic diseases. However, siRNA instability and biodistribution issues still need to be overcome, and adequate delivery vehicles have proven indispensable in conveying siRNA to its target. Chitosan is a promising biopolymer for siRNA delivery, its interest stemming from its safety, biodegradability, mucoadhesivity, permeation enhancing effect and cationic charge, as well as amenability to undergo chemical modifications. Chitosan and its derivatives can be readily arranged into complexes or nanoparticles able to entrap and carry siRNA. Specific strategies have been adopted to improve chitosan-based vectors with regard to transfectability. However, further efforts are required to verify their value and adapt them to enhance therapeutic output prior to clinical application. This review emphasizes the potential of chitosan and its derivatives to develop nanocarriers for siRNA delivery. The properties of chitosan that are significant for transfectability and the most relevant findings are assessed.
- Microencapsulation of selenium by spray-drying as a tool to improve bioaccessibility in food matrixPublication . Grenha, Ana; Guerreiro, Filipa; Lourenço, João P.; Lopes, João Almeida; Cámara-Martos, FernandoSe in the form of sodium selenite was microencapsulated by spray - drying and added to a food matrix (yogurt) to study the potential improvement of its bioaccessibility. Yogurt samples were also supplemented with Se in free salt form. Se-loaded microparticles were successfully prepared by spray-drying with production yields above 70%. The supplementation of yogurt with Se in the form of free sodium selenite had a low effect on improving the bioaccessibility of this micronutrient (1%). In turn, Se microencapsulation with mannitol or mannitol/gastro-resistant polymer (Eudragit (R)) had a strong impact on bioaccessibility results. After the gastric phase, Se bio-accessibility reached values of 21 and 40% for the microencapsulated formulations, respectively. This percentage rose to 55% at the end of intestinal phase, showing no differences between both formulations. Our results show the relevance of microencapsulation as an effective tool to improve the bioaccessibility of micronutrients when they are used in food supplementation.