α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration

Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO-expanded liquid organic solution, shows the great potential of this CO-based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest. α-galactosidase-A-(GLA)-loaded nano-liposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides are successfully prepared by using compressed CO. This nanoformulation shows an unprecedented increase of the GLA enzymatic activity and intracellular penetration, in comparison to the free enzyme. Moreover, these nanoconjugates lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide (Gb3), in a cellular model of Fabry disease, than that achieved by the free enzyme.We acknowledge financial support from Instituto de Salud Carlos III, through “Acciones CIBER”. The Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. The authors appreciate the financial support through the “Development of nanomedicines for enzymatic replacement therapy in Fabry disease” project, granted by the Fundació Marató TV3, BE-WELL (CTQ2013-40480-R) project granted by DGI (Spain), GenCat (2014-SGR-17) project financed by DGR (Catalunya), LIPOCELL project financed by CIBER-BBN and Praxis Pharmaceuticals, TERARMET (RTC-2014-2207-1) project financed by MEC (Spain) and excellence Grant CTS-6270 financed by “Junta de Andalucía>. The authors wish also to thank the Microscopy Service of UAB, especially Pablo Castro for the technical support in taking the Cryo-TEM images, and Mª Eugenia López Sánchez and Natalia García Aranda for their technical assistance in the in vitro activity assays. AV is recipient of an ICREA Academia (Generalitat de Catalunya) award. We acknowledge EMBL and Electra synchrotrons for beamtime allocation, and H. Amenitsch, B. Marmilori and B. Sartori for technical support at the SAXS beamline. Access to the synchrotron facility is supported by the BioStructX program (nº BIOSTRUCTX_1093). We also acknowledge computer time at the Minotauro-BSC supercomputer from the Red Española de Supercomputación (RES).Peer Reviewe

Experiència de flexibilitat metodològica en el procés d'ensenyament-aprenentatge. El cas de l'assignatura del grau de Farmàcia 'Biomembranes: estudis fisicoquímics'

Es descriu l'experiència realitzada en l'assignatura optativa Biomembranes: estudis fisicoquímics del Grau de Farmàcia de la UB. Aquest aspecte comporta formar un grup no molt nombrós d'estudiants i permet realitzar experiències metodològiques noves introduint aspectes de flexibilitat per a estudiants i professors. Es van establir els objectius competencials i es va dissenyar un programa flexible respecte a continguts, a activitats i dimensió espai-temps amb resultats satisfactoris

DELOS Nanovesicles-Based Hydrogels: An Advanced Formulation for Topical Use

Topical delivery has received great attention due to its localized drug delivery, its patient compliance, and its low risk for side effects. Recent developments have focused on studying new drug delivery systems as a strategy for addressing the challenges of current topical treatments. Here we describe the advances on an innovative drug delivery platform called DELOS nanovesicles for topical drug delivery. Previously, the production of DELOS nanovesicles demonstrated potentiality for the topical treatment of complex wounds, achieving well-tolerated liquid dispersions by this route. Here, research efforts have been focused on designing these nanocarriers with the best skin tolerability to be applied even to damaged skin, and on exploring the feasibility of adapting the colloidal dispersions to a more suitable dosage form for topical application. Accordingly, these drug delivery systems have been efficiently evolved to a hydrogel using MethocelTM K4M, presenting proper stability and rheological properties. Further, the integrity of these nanocarriers when being gellified has been confirmed by cryo-transmission electron microscopy and by Förster resonance energy transfer analysis with fluorescent-labeled DELOS nanovesicles, which is a crucial characterization not widely reported in the literature. Additionally, in vitro experiments have shown that recombinant human Epidermal Growth Factor (rhEGF) protein integrated into gellified DELOS nanovesicles exhibits an enhanced bioactivity compared to the liquid form. Therefore, these studies suggest that such a drug delivery system is maintained unaltered when hydrogellified, becoming the DELOS nanovesicles-based hydrogels, an advanced formulation for topical use.This research was funded by Industrial Doctorates Plan of Agaur-Generalitat de Catalunya (2018 DI 057). This work was also co-financed by European Regional Development Funds from the European Union within the framework of the operative FEDER program in Catalonia 2014–2020, by the specific project NANONAFRES (COMRDI15-1-0023). We thank the Spanish Ministry of Science and Innovation (MICINN) for financing MOL4BIO project through grant No. PID2019-105622RB-I00. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 953110. ICMAB-CSIC members acknowledge support from the MINECO through the Severo Ochoa Program for Centers of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S), and through the State Program for R&D&i (2016), orientated to the Society Challenges, modality RETOS-Collaboration 2016, and co-financed by FEDER funds from the European Commission (grant No. RTC-2016-4567-1, Nano4Derm). ICMAB-CSIC members also thank Agaur agency of Generalitat de Catalunya for their support through grant No. 2017-SGR-918. DELOS nanovesicle production and their physicochemical characterization was performed by the ICTS “NANBIOSIS”, more specifically in the Biomaterial Processing and Nanostructuring Unit (U6), Unit of the CIBER in Bioengineering, Biomaterials and Nanomedicne (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). This work has been done in the framework of the L.B.-H. doctorate in Materials Science of the Universitat Autonoma de Barcelona.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing

European “Smart-4-Fabry” project to develop a new nanomedicine for the treatment of Fabry disease

El projecte europeu «Smart-4-Fabry» té com a objectiu principal desenvolupar una nova nanomedicina, basada en l’encapsulació de l’enzim α-galactosidasa A en nanoliposomes, per al tractament de la malaltia minoritària de Fabry. El projecte parteix d’una primera prova de concepte que dona suport a aquesta estratègia basada en la nanotecnologia, i té com a repte avançar amb la seva optimització fins al final de la preclínica reguladora. Aquest article presenta d’una manera general en què consisteix i com està organitzat el projecte a fi d’aconseguir aquesta fita. Per assolir reeixidament aquest objectiu, és necessària una estreta col·laboració entre equips de recerca de diferents disciplines científiques.Paraules clau: Liposomes, nanomedicina, malaltia de Fabry, col·laboració interdisciplinària, desenvolupament de fàrmacs.The European “Smart-4-Fabry” project aims to develop a new nanoformulation based on the encapsulation of the α-galactosidase A enzyme in nanoliposomes, to improve the current treatment of Fabry disease. The project will advance from the nano-GLA from a preliminary concept test to the end of the preclinical regulatory phase. This article presents this project and explains how it is organized to achieve its goal, a process that will require a strong multidisciplinarity and cooperation between the participating research groups.Keywords: Liposomes, nanomedicine, Fabry disease, interdisciplinary collaboration, drug development

La formació del professorat novell: clau de volta pel futur de la innovació docent a la universitat dins de l'EEES

Podeu consultar la Setena trobada de professorat de Ciències de la Salut completa a: http://hdl.handle.net/2445/43352Para conseguir una docencia de excelencia y calidad en la Universidad se requiere de una preparación por parte del profesorado que la debe impartir. Si esta formación comienza desde los primeros escalones de la Universidad, los profesores noveles, ésta les permitirá alcanzar unas competencias pedagógicas básicas que podrán ir ampliando y mejorando en el transcurso de su trayectoria docente

Liposomal formulations for treating lysosomal storage disorders

Lysosomal storage disorders (LSD) are a group of rare life-threatening diseases caused by a lysosomal dysfunction, usually due to the lack of a single enzyme required for the metabolism of macromolecules, which leads to a lysosomal accumulation of specific substrates, resulting in severe disease manifestations and early death. There is currently no definitive cure for LSD, and despite the approval of certain therapies, their effectiveness is limited. Therefore, an appropriate nanocarrier could help improve the efficacy of some of these therapies. Liposomes show excellent properties as drug carriers, because they can entrap active therapeutic compounds offering protection, biocompatibility, and selectivity. Here, we discuss the potential of liposomes for LSD treatment and conduct a detailed analysis of promising liposomal formulations still in the preclinical development stage from various perspectives, including treatment strategy, manufacturing, characterization, and future directions for implementing liposomal formulations for LSD.This work has received funding from: Grant Severo Ochoa CEX2019-000917-S funded by Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación MCIN/AEI/10.13039/501100011033; Project MOL4BIO PID2019-105622RB-I00 funded by Agencia Estatal de Investigación - Ministerio de Ciencia e Innovación MCIN/AEI/10.13039/501100011033; Project PHOENIX-OITB funded by European Union’s Horizon 2020 research and innovation programme under grant agreement No 953110; Project SMART4FABRY funded by European Union’s Horizon 2020 research and innovation programme under grant agreement No 720942. The authors are also grateful to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

DELOS nanovesicles-based hydrogels: An innovative formulation for topical use

L’alliberament de fàrmacs per a ús tòpic ha rebut una gran atenció pel fet que es tracta d’un alliberament localitzat, per l’alta acceptació per part del pacient i pel seu baix risc d’efectes secundaris. Aquest article descriu els avenços d’una plataforma innovadora d’alliberament de fàrmacs anomenada nanovesícules DELOS per a ús tòpic. Concretament, aquestes nanovesícules s’han transformat en hidrogel d’una manera eficient i assegurant-ne la integritat, i s’han convertit així en una formulació avançada per a ús tòpic.Topical delivery has received great attention due to its localized drug delivery effect, its patient compliance, and its low risk of side effects. This article describes the advances in an innovative drug delivery platform called DELOS nanovesicles for topical drug delivery. Specifically, these nanovesicles have been efficiently transformed into hydrogels, ensuring the integrity of the vesicles and making them an advanced formulation for topical use

Experiència de flexibilitat metodològica en el procés d'ensenyament-aprenentatge. El cas de l'assignatura del grau de Farmàcia 'Biomembranes: estudis fisicoquímics'

Es descriu l'experiència realitzada en l'assignatura optativa Biomembranes: estudis fisicoquímics del Grau de Farmàcia de la UB. Aquest aspecte comporta formar un grup no molt nombrós d'estudiants i permet realitzar experiències metodològiques noves introduint aspectes de flexibilitat per a estudiants i professors. Es van establir els objectius competencials i es va dissenyar un programa flexible respecte a continguts, a activitats i dimensió espai-temps amb resultats satisfactoris

MKC-Quatsomes: a stable nanovesicle platform for bio-imaging and drug-delivery applications

Quatsomes are outstanding new lipid-based nanovesicles that are highly homogeneous and stable in different media for years, but the composition must be carefully chosen to avoid any potentially toxic side effects in in vivo applications. To this end, we have developed and studied a novel type of Quatsomes composed of cholesterol and myristalkonium chloride (MKC), the latter being extensively used as antimicrobial preservative in many ophthalmic and parenteral formulations on the EU and USA market. We have synthesized these novel MKC-Quatsomes in different media that are suitable for parenteral administration, and confirmed their stability in these media for 18 months, as well as the stability in human serum for 24 hours. Biodistribution assays were performed after intravenous injection of fluorescently labeled MKC-Quatsomes in live mice bearing xenografted colorectal tumors, showing nanovesicle accumulation in tumors, liver, spleen, and kidneys. No histological alteration or toxicity was observed in any of these organs.This work is financially supported by the Ministry of Economy, Industry and Competitiveness, Spain, through the “MOTHER” project (MAT2016-80826-R). The authors also thank the “FLOWERS” (FUNMAT-FIP-2016) project funded by the Severo Ochoa (SEV-2015-0496) awarded to ICMAB by the Ministry of Economy and Competitiveness. The authors acknowledge financial support from Instituto de Salud Carlos III, through “Acciones CIBER”. The Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) is funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Authors also acknowledge the NANOMETS project, funded by CIBER-BBN call Intramurales 2016-2017. The production and physico-chemical characterization of MKC-quatsomes have been performed at the ‘Biomaterial Processing and Nanostructuring Unit (U6) of CIBER-BBN of ICTS “NANBIOSIS”, located at ICMAB-CSIC. In vivo toxicology and biodistribution studies were performed at the Nanotoxicology Unit (U18) of CIBER-BBN located at the Hospital de la Santa Creu i Sant Pau. The authors wish to thank Dr. Iván López-Montero (Physical Chemistry, Universidad Complutense de Madrid) for fluorescence confocal microscopy measurements.Peer reviewe