Procedimiento para la obtención de micro- o nanopartículas sólidas

Procedimiento para la obtencion de micro- o nanoparticulas solidas. La invencion proporciona un nuevo procedimiento para la obtencion de micro- o nanoparticulas solidas con estructura homogenea. Se proporciona un procedimiento que permite obtener micro- o nanoparticulas solidas de estructura homogenea, con un tamano de particula inferior a 10 ƒÊm donde el compuesto solido procesado revela la naturaleza, cristalina, amorfa, polimorfica, etc..., propia del compuesto de partida. De acuerdo con la invencion se proporciona un procedimiento que ademas permite obtener micro- o nanoparticulas solidas con una morfologia sustancialmente esferoidal.Peer reviewedConsejo Superior de Investigaciones Científicas (Esspaña), Centro de Investigación Biomédica en Red en Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN)A1 Solicitud de patente con informe sobre el estado de la técnic

Controlled crystallization of Mn12 single-molecule magnets by compressed CO2 and its influence on the magnetization relaxation

6 pages, 6 figures, 2 tables.Micro- and sub-micro particles of complex [Mn12O12(O2CC6H5)16(H2O)4] ( 1) with controlled size and polymorphism have been prepared by dense-gas crystallization techniques, showing a remarkable particle size influence on the magnetization relaxation rates.This work was supported by DGI (Spain) under projects MAT2002-0043 and MAT2003-04699 and by the European Commission under the NoE MAGMANET (Contract NMP3- CT-2005-515767) and QUEMOLNA Marie Curie RTN (Contract MRTN-CT-2003-5044880). Javier Campo and Nora Ventosa thank the Ramon y Cajal Program of Ministerio de Educación y Tecnología (Spain) for their contracts. Maria Muntó thanks the Consejo Superior de Investigaciones Científicas (CSIC) for her PhD bursary and Jordi Gómez- Segura thanks the European Community for his PhD grant.Peer reviewe

Lipid-based nanovesicles for nanomedicine

Molecular self-assembly has enabled the fabrication of biologically inspired, advanced nanostructures as lipid-based nanovesicles (L-NVs). The oldest L-NVs, liposomes, have been widely proposed as potential candidates for drug delivery, diagnostic and/or theranostic applications and some liposome-based drug products have already stepped from the lab-bench to the market. This success is attributed to their ability to encapsulate both hydrophobic and/or hydrophilic molecules, efficiently carry and protect them within the body and finally deliver them at the target site. These positive features are also coupled with high biocompatibility. However, liposomes still present some unsolved drawbacks, such as poor colloidal stability, short shelf-life, restricted and expensive conditions of preparation because of the inherent nature of their fundamental constituents (phospholipids). The new tools available in the self-assembly of controlled molecules have significantly advanced the field of L-NV design and synthesis, and nonliposomal L-NVs have been recently developed; this new generation of nanovesicles can represent a paradigm shift in nanomedicine: they may complement liposomes, showing their advantages and overcoming most of their drawbacks. Clearly, being still young, their rocky way to the clinic first and then to the market has just started and it is still long, but they have all the potentialities to reach their objective target. The purpose of this review is to first present the large plethora of L-NVs available, focusing on this new generation of non-liposomal L-NVs and showing their similarities and differences with respect to their ancestors (liposomes). Since the overspread of a nanomaterial to the market is also strongly dependent on the availability of technological-scale preparation methods, we will also extensively review the current approaches exploited for L-NV production. The most cutting-edge approaches based on compressed fluid (CF) technologies will be highlighted here since they show the potential to represent a game-change in the production of L-NVs, favouring their step from the bench to the market. Finally, we will briefly discuss L-NV applications in nanomedicine, looking also for their future perspectives.This work was financially supported by MINECO (DGI), Spain, grants BE-WELL CTQ2013-40480-R, TERARMET (RTC-2014-2207-1), QUATFORFRAG (RTC-2014-2183-5) and UNDERLIPIDS (RTC-2015-3303-1), and by AGAUR, Generalitat de Catalunya, “Grant 2014-SGR-17“. N.G. acknowledges the European Commission (EC) (FP7-PEOPLE-2013-Initial Training Networks (ITN) ‘‘NANO2FUN’’ Project No. 607721) for their Postdoctoral contract. The authors appreciate support from LIPOCELL project financed by CIBER-BBN and Praxis Pharmaceuticals and also acknowledge the 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.Peer reviewe

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

Crystal size dependence of dipolar ferromagnetic order between Mn6 molecular nanomagnets

We study how crystal size influences magnetic ordering in arrays of molecular nanomagnets coupled by dipolar interactions. Compressed fluid techniques have been applied to synthesize crystals of Mn6 molecules (spin S=12) with sizes ranging from 28μm down to 220 nm. The onset of ferromagnetic order and the spin thermalization rates have been studied by means of ac susceptibility measurements. We find that the ordered phase remains ferromagnetic, as in the bulk, but the critical temperature Tc decreases with crystal size. Simple magnetostatic energy calculations, supported by Monte Carlo simulations, account for the observed drop in Tc in terms of the minimum attainable energy for finite-sized magnetic domains limited by the crystal boundaries. Frequency-dependent susceptibility measurements give access to the spin dynamics. Although magnetic relaxation remains dominated by individual spin flips, the onset of magnetic order leads to very long spin thermalization time scales. The results show that size influences the magnetism of dipolar systems with as many as 1011 spins and are relevant for the interpretation of quantum simulations performed on finite lattices

Novel bioactive hydrophobic gentamicin carriers for the treatment of intracellular bacterial infections.

Gentamicin (GEN) is an aminoglycoside antibiotic with a potent antibacterial activity against a wide variety of bacteria. However, its poor cellular penetration limits its use in the treatment of infections caused by intracellular pathogens. One potential strategy to overcome this problem is the use of particulate carriers that can target the intracellular sites of infection. In this study GEN was ion paired with the anionic AOT surfactant to obtain a hydrophobic complex (GEN-AOT) that was formulated as a particulated material either by the Precipitation with a Compressed Antisolvent (PCA) method, or by encapsulation into poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The micronization of GEN-AOT by PCA yielded a particulated material with a higher surface area than the non-precipitated complex, while PLGA NPs within a size range of 250-330 nm and a sustained release of the drug over 70 days were obtained by preparing the NPs using the emulsion solvent evaporation method. For the first time, GEN encapsulation efficiency values around 100% were achieved for the different NP formulations with no signs of interaction between the drug and the polymer. Finally, in vitro studies against the intracellular bacteria Brucella melitensis, used as a model of intracellular pathogen, demonstrated that the bactericidal activity of GEN was unmodified after ion-pairing, precipitation or encapsulation into NPs. These results, encourage their use for treatment for infections caused by GEN sensitive intracellular bacteria

Cellular pharmacokinetics and intracellular activity against Listeria monocytogenes and Staphylococcus aureus of chemically modified and nanoencapsulated gentamicin

OBJECTIVES: The aim of this study was to investigate different hydrophobic gentamicin formulations [gentamicin-bis(2-ethylhexyl) sulfosuccinate (GEN-AOT), microstructured GEN-AOT (PCA GEN-AOT) and GEN-AOT-loaded poly(lactide-co-glycolide) acid (PLGA) nanoparticles (NPs)] in view of improving its therapeutic index against intracellular bacteria. The intracellular accumulation, subcellular distribution and intracellular activity of GEN-AOT and NPs in different monocytic-macrophagic cell lines were studied. METHODS: Human THP-1 and murine J774 phagocytic cells were incubated with GEN-AOT formulations at relevant extracellular concentrations [from 1× MIC to 18 mg/L (human C(max))], and their intracellular accumulation, subcellular distribution and toxicity were evaluated and compared with those of conventional unmodified gentamicin. Intracellular activity of the formulations was determined against bacteria showing different subcellular localizations, namely Staphylococcus aureus (phagolysosomes) and Listeria monocytogenes (cytosol). RESULTS: GEN-AOT formulations accumulated 2-fold (GEN-AOT) to 8-fold (GEN-AOT NPs) more than gentamicin in phagocytic cells, with a predominant subcellular localization in the soluble fraction (cytosol) and with no significant cellular toxicity. NP formulations allowed gentamicin to exert its intracellular activity after shorter incubation times and/or at lower concentrations. With an extracellular concentration of 10× MIC, a 1 log(10) decrease in S. aureus intracellular inoculum was obtained after 12 h instead of 24 h for NPs versus free gentamicin, and a static effect was observed against L. monocytogenes at 24 h with NPs, while free gentamicin was ineffective. CONCLUSIONS: GEN-AOT formulations yielded a high cellular accumulation, especially in the cytosol, which resulted in improved efficacy against both intracellular S. aureus and L. monocytogenes

Quatsomes Loaded with Squaraine Dye as an Effective Photosensitizer for Photodynamic Therapy

Photodynamic therapy is a non-invasive therapeutic strategy that combines external light with a photosensitizer (PS) to destroy abnormal cells. Despite the great progress in the development of new photosensitizers with improved efficacy, the PS’s photosensitivity, high hydrophobicity, and tumor target avidity still represent the main challenges. Herein, newly synthesized brominated squaraine, exhibiting intense absorption in the red/near-infrared region, has been successfully incorporated into Quatsome (QS) nanovesicles at different loadings. The formulations under study have been characterized and interrogated in vitro for cytotoxicity, cellular uptake, and PDT efficiency in a breast cancer cell line. The nanoencapsulation of brominated squaraine into QS overcomes the non-water solubility limitation of the brominated squaraine without compromising its ability to generate ROS rapidly. In addition, PDT effectiveness is maximized due to the highly localized PS loadings in the QS. This strategy allows using a therapeutic squaraine concentration that is 100 times lower than the concentration of free squaraine usually employed in PDT. Taken together, our results reveal the benefits of the incorporation of brominated squaraine into QS to optimize their photoactive properties and support their applicability as photosensitizer agents for PDT

Ultrabright Föster Resonance Energy Transfer Nanovesicles:The Role of Dye Diffusion

The development of contrast agents based on fluorescent nanoparticles with high brightness and stability is a key factor to improve the resolution and signal-to-noise ratio of current fluorescence imaging techniques. However, the design of bright fluorescent nanoparticles remains challenging due to fluorescence self-quenching at high concentrations. Developing bright nanoparticles showing FRET emission adds several advantages to the system, including an amplified Stokes shift, the possibility of ratiometric measurements, and of verifying the nanoparticle stability. Herein, we have developed Förster resonance energy transfer (FRET)-based nanovesicles at different dye loadings and investigated them through complementary experimental techniques, including conventional fluorescence spectroscopy and super-resolution microscopy supported by molecular dynamics calculations. We show that the optical properties can be modulated by dye loading at the nanoscopic level due to the dye's molecular diffusion in fluid-like membranes. This work shows the first proof of a FRET pair dye's dynamism in liquid-like membranes, resulting in optimized nanoprobes that are 120-fold brighter than QDot 605 and exhibit >80% FRET efficiency with vesicle-to-vesicle variations that are mostly below 10%.J.M.-F. gratefully thanks the financial support received by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 712949 (TECNIOspring PLUS) and from the Agency for Business Competitiveness of the Government of Catalonia. We acknowledge the European Commission (EC) FP7-PEOPLE-2013-Initial Training Networks (ITN) “NANO2FUN” project no. 607721 for being the spark that initiates this work and EC project MSCA-RISE-2020 "MICRO4NANO" project no.101007804. This work was also financially supported by Generalitat de Catalunya (grant no. 2017-SGR-918), the Ministry of Economy, Industry, and Competitiveness (Spain), through the “MOTHER” project (MAT2016-80826-R), the Ministry of Science and Innovation of Spain through the grant PID2019-105622RB-I00 (Mol4Bio). ICMAB-CSIC also acknowledges support from the MINECO through the Severo Ochoa Programme FUNFUTURE (SEV-2015-0496 and CEX2019-000917-S). K.D.B. acknowledges the National Science Foundation (CBET-1517273 and CHE-1726345). C.S. and A.P. benefited from the equipment and framework of the COMP-HUB Initiative, funded by the “Departments of Excellence” program of the Italian Ministry for Education, University and Research (MIUR, 2018-2022). We thank the CESGA Supercomputing Center for technical support and the use of computational resources. The contribution of S.I.-T. has been done under the Materials Science PhD program in the Barcelona Autonomous University (UAB). Characterizations of nanovesicles were made at the ICTS “NANBIOSIS”, more specifically by the U6 unit of CIBER-BBN. The authors would like also to thank the collaboration of Hamamatsu Photonics for the quantum yield determinations using the Quantaurus-QY Plus UV–NIR absolute PL quantum yield spectrometer.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe