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Bioorthogonal metallocatalysis has opened up a xenobiotic route to perform nonenzymatic catalytic transformations in living settings. Despite their promising features, most metals are deactivated inside cells by a myriad of reactive biomolecules, including biogenic thiols, thereby limiting the catalytic functioning of these abiotic reagents. Here we report the development of cytocompatible alloyed AuPd nanoparticles with the capacity to elicit bioorthogonal depropargylations with high efficiency in biological media. We also show that the intracellular catalytic performance of these nanoalloys is significantly enhanced by protecting them following two different encapsulation methods. Encapsulation in mesoporous silica nanorods resulted in augmented catalyst reactivity, whereas the use of a biodegradable PLGA matrix increased nanoalloy delivery across the cell membrane. The functional potential of encapsulated AuPd was demonstrated by releasing the potent chemotherapy drug paclitaxel inside cancer cells. Nanoalloy encapsulation provides a novel methodology to develop nanoreactors capable of mediating new-to-life reactions in cells., UK Research & Innovation (UKRI), Engineering & Physical Sciences Research Council (EPSRC) EP/N021134/1, ERC (Advanced Grant CADENCE) ERC-2016-ADG-742684, European Commission European Commission Joint Research Centre H2020-MSCA-IF-2014-658833 H2020-MSCA-IF-2018-841990 H2020-MSCA-IF-2016-749299 H2020-MSCA-IF-2019-895664, Spanish Government RTI2018-099019-A-I00

Mesenchymal stem cells have contributed to the continuous progress of tissue engineering and regenerative medicine. Adipose-derived stem cells (ADSC) possess many advantages compared to other origins including easy tissue harvesting, self-renewal potential, and fast population doubling time. As multipotent cells, they can differentiate into osteoblastic cell linages. In vitro bone models are needed to carry out an initial safety assessment in the study of novel bone regeneration therapies. We hypothesized that 3D bone-on-a-chip models containing ADSC could closely recreate the physiological bone microenvironment and promote differentiation. They represent an intermedium step between traditional 2D–in vitro and in vivo experiments facilitating the screening of therapeutic molecules while saving resources. Herein, we have differentiated ADSC for 7 and 14 days and used them to fabricate in vitro bone models by embedding the pre-differentiated cells in a 3D collagen matrix placed in a microfluidic chip. Osteogenic markers such as alkaline phosphatase activity, calcium mineralization, changes on cell morphology, and expression of specific proteins (bone sialoprotein 2, dentin matrix acidic phosphoprotein-1, and osteocalcin) were evaluated to determine cell differentiation potential and evolution. This is the first miniaturized 3D-in vitro bone model created from pre-differentiated ADSC embedded in a hydrogel collagen matrix which could be used for personalized bone tissue engineering.

Osteoarthritis (OA) is a common chronic joint pathology that has become a predominant cause of disability worldwide. Even though the origin and evolution of OA rely on different factors that are not yet elucidated nor understood, the development of novel strategies to treat OA has emerged in the last years. Cartilage degradation is the main hallmark of the pathology though alterations in bone and synovial inflammation, among other comorbidities, are also involved during OA progression. From a molecular point of view, a vast amount of signaling pathways are implicated in the progression of the disease, opening up a wide plethora of targets to attenuate or even halt OA. The main purpose of this review is to shed light on the recent strategies published based on nanotechnology for the early diagnosis of the disease as well as the most promising nano-enabling therapeutic approaches validated in preclinical models. To address the clinical issue, the key pathways involved in OA initiation and progression are described as the main potential targets for OA prevention and early treatment. Furthermore, an overview of current therapeutic strategies is depicted. Finally, to solve the drawbacks of current treatments, nanobiomedicine has shown demonstrated benefits when using drug delivery systems compared with the administration of the equivalent doses of the free drugs and the potential of disease-modifying OA drugs when using nanosystems. We anticipate that the development of smart and specific bioresponsive and biocompatible nanosystems will provide a solid and promising basis for effective OA early diagnosis and treatment., This work was supported by the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III) and the Proyectos de Investigación en Salud (AES 2021) Program (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación). M.P. acknowledges the support from the DGA fellowship (Orden CUS/581/2020) granted by the Regional Government of Aragon. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund.

The antimicrobial action of gold depends on different factors including its oxidation state in the intra- and extracellular medium, the redox potential, its ability to produce reactive oxygen species (ROS), the medium components, the properties of the targeted bacteria wall, its penetration in the bacterial cytosol, the cell membrane potential, and its interaction with intracellular components. We demonstrate that different gold species are able to induce bacterial wall damage as a result of their electrostatic interaction with the cell membrane, the promotion of ROS generation, and the consequent DNA damage. In-depth genomic and proteomic studies on Escherichia coli confirmed the superior toxicity of Au (III) vs Au (I) based on the different molecular mechanisms analyzed including oxidative stress, bacterial energetic metabolism, biosynthetic processes, and cell transport. At equivalent bactericidal doses of Au (III) and Au (I) eukaryotic cells were not as affected as bacteria did, maintaining unaffected cell viability, morphology, and focal adhesions; however, increased ROS generation and disruption in the mitochondrial membrane potential were also observed. Herein, we shed light on the antimicrobial mechanisms of ionic and biogenic gold nanoparticles against bacteria. Under selected conditions antibiotic-like ionic gold can exert a strong antimicrobial activity while being harmless to human cells., Financial support from the Spanish Ministry of Science and Innovation (grant numbers PID2020-113987RB-I00, PID2021-127847OB-I00 and CTQ2017-84473-R) is gratefully acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund. We acknowledge the ELECMI-LMA-INA (University of Zaragoza, Spain), Nanbiosis ICTS, the Genomics Core Unit from Instituto de Investigaciones Biomédicas Alberto Sols (Madrid, Spain), and Cell Culture, Cell Separation and Flow Cytometry, Genomics and Proteomics Core Units from IACS/IIS Aragon for facilitating the access to their instruments and expertise. We would like to thank Dr. Gema Rodríguez-Tarduchy for helpful advice regarding genomics studies. M.P. and C.R. acknowledge the support from Aragon regional government (Orden CUS/581/2020 and Orden CUS/803/2021, respectively). G.M. gratefully acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III).

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Compared to parenteral administration of colistin, its direct pulmonary administration can maximize lung drug deposition while reducing systemic adverse side effects and derived nephrotoxicity. Current pulmonary administration of colistin is carried out by the aerosolization of a prodrug, colistin methanesulfonate (CMS), which must be hydrolized to colistin in the lung to produce its bactericidal effect. However, this conversion is slow relative to the rate of absorption of CMS, and thus only 1.4 % (w/w) of the CMS dose is converted to colistin in the lungs of patients receiving inhaled CMS. We synthesized several aerosolizable nanoparticle carriers loaded with colistin using different techniques and selected particles with sufficient drug loading and adequate aerodynamic behavior to efficiently deliver colistin to the entire lung. Specifically, we carried out (i) the encapsulation of colistin by single emulsion-solvent evaporation with immiscible solvents using polylactic-co-glycolic (PLGA) nanoparticles; (ii) its encapsulation using nanoprecipitation with miscible solvents using poly(lactide-co-glycolide)-block-poly(ethylene glycol) as encapsulating matrix; (iii) colistin nanoprecipitation using the antisolvent precipitation method and its subsequent encapsulation within PLGA nanoparticles; and (iv) colistin encapsulation within PLGA-based microparticles using electrospraying. Nanoprecipitation of pure colistin using antisolvent precipitation showed the highest drug loading (55.0 ± 4.8 wt%) and spontaneously formed aggregates with adequate aerodynamic diameter (between 3 and 5 μm) to potentially reach the entire lung. These nanoparticles were able to completely eradicate Pseudomonas aeruginosa in an in vitro lung biofilm model at 10 µg/mL (MBC). This formulation could be a promising alternative for the treatment of pulmonary infections improving lung deposition and, therefore, the efficacy of aerosolized antibiotics., Financial support from the Spanish Ministry of Science and Innovation (grant numbers PID2020-113987RB-I00, CTQ2017-84473-R and PID2021-127847OB-I00) is gratefully acknowledged. This manuscript is the result of the projects PDC2021-121405-I00 and PDC2022-133866-I00, founded by MCIN/AEI/10.13039/501100011033 and by the European Union “NextGenerationEU”/PRTR. The regional Governments of Aragon and Nouvelle-Aquitaine (by means of the Order PRE/2106/2017 and arrêté n°19000160 EMPIRE project) are also gratefully acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund.

This work tackles the quest for temperature-responsive greener solvents by synthesizing a hydrophobic deep eutectic solvent (DES) comprising menthol and decanoic acid. The low solubility of hydroph...

Copolymers synthesized from acrylic acid and methacrylic acid used as gastroprotective and mucoadhesive enteric coatings have been used to prepare micro- (∼2 μm), submicro- (∼200 nm), and nanoparticles (∼20 nm) containing rifampicin (Rif) to obtain time-controlled drug release kinetics. Different particle sizes and drug release kinetics have been obtained using different synthesis conditions and fabrication techniques including the use of an electrosprayer and an interdigital microfabricated micromixer. The antimicrobial action of the encapsulated Rif has been demonstrated against Staphylococcus aureus ATCC 25923 and compared with the effect of the equivalent dose of the free macrolide antibiotic. At low concentrations, the encapsulated antibiotic showed superior antimicrobial activity than the free drug. The stability of the developed particles has been evaluated in vitro under simulated gastric and intestinal conditions. At the concentrations tested, a reduced cytotoxicity against different human cell lines was observed after analyzing their subcytotoxic doses and the influence on their cell cycle by flow cytometry. Drug release kinetics can be tuned by adjusting particle sizes, and it would be possible to reach the minimum inhibitory concentration or the minimum bactericidal concentration at different time points depending on the medical needs., This research was funded by the Spanish Ministry of Economy and Competitiveness (grant number CTQ2017-84473-R). Financial support from the EU thanks to the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM) is also acknowledged. 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 (Spain) with assistance from the European Regional Development Fund. V.S. acknowledges the financial support of Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00.

[Aim]: First, to compare in vitro minimum inhibitory concentrations (MIC) of free cloxacillin and cloxacillin-containing nanoparticles (NP) against methicillin-susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) and second, to assess NP antimicrobial activity against intracellular S. aureus., [Methods]: Poly(d,l-lactide-co-glycolide) acid (PLGA)-NP were loaded with cloxacillin and physico-chemically characterized. MICs were determined for reference strains Newman-(MSSA) and USA300-(MRSA). Murine alveolar macrophages were infected, and bacterial intracellular survival was assessed after incubating with free-cloxacillin or PLGA-cloxacillin-NP., [Results and conclusion]: For both isolates, MICs for antibiotic-loaded-NP were lower than those obtained with free cloxacillin, indicating that the drug encapsulation improves antimicrobial activity. A sustained antibiotic release was demonstrated when using the PLGA-cloxacillin-NP. When considering the lowest concentrations, the use of drug-loaded NP enabled a higher reduction of intracellular bacterial load., This project was funded jointly by SEPAR (Spanish Society for Pneumology and Thoracic Surgery), CIBER-BBN and CIBERES (Spanish Consortia for Research on Bioengineering, Biomaterials and Nanomedicine, and on Respiratory Diseases, respectively). This research was also supported by the Instituto de Salud Carlos III (PI 17/01139), integrated in the Plan Nacional de I+D+I and co-funded jointly by the ISCIII Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). The authors also thank the support from The Spanish Ministry of Science, Innovation and Universities (grant: CTQ2017-84473-R). We also acknowledge financial support from the EU (ERC Consolidator grant program, ERC-2013-CoG-614715, NANOHEDONISM). J Domínguez and G Mendoza are researchers from the Miguel Servet Program. C Prat was awarded by Programa Germans Trias Sapiens-Fundació Catalunya la Pedrera and European Respiratory Society – ERS Short-Term Research Fellowship 2018 (STRTF201810-00467).

Due to the prevalence of antimicrobial resistant pathogens, natural products with long-term antimicrobial activities are considered as potential alternatives. In this work, polycaprolactone (PCL) electrospun fibers with mean diameters around 299 nm and loaded with 14.92 ± 1.31% w/w thymol (THY) were synthesized. The mats had appropriate elongation at break (74.4 ± 9.5%) and tensile strength (3.0 ± 0.5 MPa) to be potentially used as wound dressing materials. In vivo studies were performed using eight to ten week-old male SKH1 hairless mice. The infection progression was evaluated through a semiquantitative method and quantitative polymerase chain reaction. The analyses of post-mortem samples indicated that THY-loaded PCL fibers acted as inhibitors of Staphylococcus aureus ATCC 25923 strain growth being as efficient as chlorhexidine (CLXD). Histopathological and immunohistochemical studies showed that the PCL-THY-treated wounds were almost free of an inflammatory reaction. Therefore, wound dressings containing natural compounds can prevent infection and promote wound healing and prompt regeneration. Copyright

The acidic pH of healthy skin changes during wound healing due to the exposure of the inner dermal and subcutaneous tissue and due to the potential colonization of pathogenic bacteria. In chronic non-healing wounds, the pH values vary in a wide pH range but the appearance of an alkaline shift is common. After a wound is incurred, neutral pH in the wound bed is characteristic of the activation of the cascade of regenerative and remodeling processes. In order to adjust drug release to the specific pH of the wound, herein, drug-loaded wound dressings having pH-responsiveness containing antiseptics and antibiotics and exerting different release kinetics in order to have a perfect match between the drug release kinetics, and the pH conditions of each wound type, were developed. We have fabricated drug-loaded electrospun nanofibers loaded with the antiseptic chlorhexidine, with the broad-spectrum antibiotic rifampicin, and with the antimicrobial of natural origin thymol, using the pH-dependent methacrylic acid copolymer Eudragit® L100-55, which dissolves at pH > 5.5; those drugs were loaded within Eudragit® S100, which dissolves at pH > 7 and, finally, within the methacrylic ester copolymer Eudragit® RS100 which is pH independent and slowly erodes and releases its contained cargo. The antibacterial action of those advanced wound dressings has been evaluated against methicillin-sensitive S. aureus Newman strain expressing the coral green fluorescent protein (cGFP), as a model of a Gram-positive bacteria, and against E. coli S17 strain as a model of a Gram-negative bacteria. It was demonstrated that those combinational products integrate in one device the required characteristics for a wound dressing with the therapeutic action of a contained active principle and can be selected depending on the wound acidic or alkaline status for its appropriated management., This research was funded by the Spanish Ministry of Science and Innovation (grant number PID2020-113987RB-I00). G.L. acknowledges the support from the FPI fellowship PRE2018-085769 granted by the Spanish Ministry of Science, Innovation and Universities, while L.G.M-C. acknowledges the financial support from the Mexican Council of Science and Technology (CONACyT) through doctoral grant #710618. G.M. gratefully acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III). 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 (Spain) with assistance from the European Regional Development Fund.

The synthesis of polymeric nanoparticles from a block copolymer based on poly(ethylene glycol) and a polymethacrylate containing the nucleobase analog 2,6-diacylaminopyridine is optimized by microfluidics to obtain homogeneous spherical micelles. Loading and delivery properties are studied using naproxen as a model. The incorporation of a Pd precursor in the polymer organic solution fed into the micromixer allows the preparation of Pd(II) precursor-polymer hybrid systems and the subsequent reduction with CO leads to the in situ synthesis of Pd nanosheets inside of the hydrophobic core of the polymeric micelles. This methodology is highly efficient to yield all polymeric nanoparticles loaded with Pd nanosheets as detected by electron microscopy and energy-dispersive X-ray spectroscopy. The cell viability of these Pd nanosheets-containing polymeric nanoparticles is evaluated using five cell lines, showing a high cytocompatibility at the tested concentrations without detrimental effects in cell membrane and nuclei. Furthermore, the use of these hybrid polymeric nanoparticles as photothermal transductors is evaluated using near infrared as irradiation source as well as its application in photothermal therapy using different cell lines demonstrating a high efficiency in all cell cultures., This work was supported by the “Ministerio de Economía y Competitividad, Programa Excelencia” (MINECO)-FEDER, Spain, under the project grant number MAT2017-84838-P (L.O. and M.P.) and by “Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación,” grant number: RTI2018-099019-A-I00 (V.S.), “Fondo Social Europeo and Gobierno de Aragón”-"Fondo Europeo de Desarrollo Regional", FEDER (E47_17R, FEDER 2014–2020 “Construyendo Europa desde Aragón”). M.A. acknowledges Gobierno de Aragón and Fondo Social Europeo for her Ph.D. grant. G.M. is also grateful for the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III).

Stringent regulations and growing industrial interests have ensued the development of sustainable processes. Membrane assisted-nanoemulsification based on microengineered membranes has been explored for the sustainable production of therapeutic hydrophobic deep eutectic solvent (DES)-based nanoemulsions for enhanced antimicrobial applications. Hydrophobic DESs were synthesised by employing therapeutic terpenes: DL-menthol and thymol. To understand the mechanism of oil droplet detachment from the membrane when producing such nanoemulsions, the effect of hydrophilicity/hydrophobicity partition of the active membrane surface on the process performance was evaluated. Using a continuous phase cross-flow velocity of 0.32 m.s−1, a dispersed phase flowrate of 0.02 ml.min−1 and 2% (w/w) Tween 80 resulted in a DES-in-water nanoemulsion of 81.2 ± 0.9 nm droplet size. Novel observations like intrinsically reduced interfacial tension of 7.5 mN.m−1 between water and hydrophobic DES, and a hydrophobic DES wetting behaviour to both hydrophilic and hydrophobic active membrane surfaces were witnessed. Compared to DL-menthol, thymol or synthesised DES, the optimised DES-in-water nanoemulsions exhibited enhanced synergetic antimicrobial effect against Escherichia coli, Staphylococcus aureus and superior potency against Cutibacterium acnes, an acne inducing bacterial strain. These nanoemulsions were also cytocompatible with human keratinocytes and dermal fibroblasts. Lastly, membrane-assisted nanoemulsification manifested controlled size and monomodally distributed nanoemulsions compared to traditional ultrasound-driven emulsification., The authors would like to acknowledge Executive Agency for Education, Audiovisual & Culture (EACEA) of the European Commission for the scholarship grant of Erasmus Mundus Doctorate in Membrane Engineering (EUDIME) program to Syed Usman Taqui. Prof. Reyes Mallada and Dr. Ruth Lahoz from Nanoscience Institute of Aragon (INA), University of Zaragoza are acknowledged for their unconditional support in fabrication and characterization of metallic membranes. This work is also supported by the Associate Laboratory for Green Chemistry - LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020). Authors M.T.B. Crespo, F.B. Gaspar and I.C. Leonardo acknowledge the financial support from the project “MobFood – Mobilizing scientific and technological knowledge in response to the challenges of the agri-food market” (POCI-01-0247-FEDER-024524) financed by European Regional Development Fund (ERDF), through the Incentive System to Research and Technological development, within the Portugal2020 Competitiveness and Internationalization Operational Program; iNOVA4Health - UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement; and INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC). This research was partially funded by the Spanish Ministry of Economy and Competitiveness (grant number CTQ2014-52384-R). V. Sebastian acknowledges the financial support from Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00. G. Mendoza gratefully acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III). 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 (Spain) with assistance from the European Regional Development Fund.

The on-demand administration of anaesthetic drugs can be a promising alternative for chronic pain management. To further improve the efficacy of drug delivery vectors, high drug loadings combined with a spatiotemporal control on the release can not only relief the pain according to patient’s needs, but also improve the drawbacks of conventional burst release delivery systems. In this study, a hybrid nanomaterial was developed by loading bupivacaine nanocrystals (BNCs) into oligo(ethylene glycol) methyl ether methacrylate (OEGMA)-based thermoresponsive nanogels and coupling them to NIR-absorbing biodegradable copper sulphide nanoparticles (CuS NPs). Those CuS NPs were surface modified with polyelectrolytes using layer-by-layer techniques to be efficiently attached to the surface of nanogels by means of supramolecular interactions. The encapsulation of bupivacaine in the form of nanocrystals allowed to achieve CuS@BNC-nanogels having drug loadings as high as 65.5 wt%. The nanocrystals acted as long-lasting drug reservoirs, leading to an elevated localized drug content, which was useful for their application in prolonged pain relief. The CuS@BNC-nanogels exhibited favorable photothermal transducing properties upon NIR-light irradiation. The photothermal effect granted by the CuS NPs triggered the nano-crystallized drug release to be boosted by the collapse of the thermoresponsive nanogels upon heating. Remote control was achieved for on-demand release at a specific time and place, indicating their potential use as an externally activated triggerable drug-delivery system. Furthermore, cell viability tests and flow cytometry analysis were performed showing satisfactory cytocompatibility in the dose-ranging study having a subcytotoxic concentration of 0.05 mg/mL for CuS@BNC-nanogels. This remotely activated nanoplatform is a promising strategy for long-lasting controlled analgesia and a potential alternative for clinical pain management., The authors thank financial support from the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM). V.S. acknowledges the financial support from Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund. Authors acknowledge the LMA-INA, Cell Separation and Flow Cytometry, Cell Culture Units from IACS/IIS Aragon (Spain) for offering access to their instruments and expertise. G.M. thanks the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III).

Many long-acting extended drug release systems can provide controlled or sustained release of therapeutic payloads. In the majority of those systems drug release cannot be stopped once it has started because they operate autonomously regardless of the evolution of the treatment and/or the patient´s needs. However, in several pathologies such as diabetes, hormonal disorders, pain management, etc. a pulsatile drug release is required to adjust the dose of drug release to the specific needs in a spatio-temporal manner. Additionally, in other pathologies such as cancer or antimicrobial therapy the release of the drug with spatio-temporal control to prevent unwanted side effects represents an unmet need. With this aim reversible stimuli-responsive nanomaterials with an on-off switching ability have been developed in order to provide a spatio-temporal control of the drug released. Those systems can be activated in response to exogenous (light, magnetic field, electrical fields, etc.) or endogenous triggers (pH, enzyme-substrate complex formation, protein-cell binding, etc.) thanks to the use of reversible phase-transition materials. In this review we compile in vitro and preclinical results in which those materials have been successfully used. The types of stimuli used to trigger drug release as well as the different nanomaterials used are reviewed in order to provide a general overview of the field. We anticipate that further studies in this field will be expanded towards the development of multimodal hybrid systems which combine therapy and imaging while reporting the evolution of the treatment in real time.

Regional anesthesia is widely used in peripheral nerve block and in neuraxial anesthesia to reduce anesthetics systemic side effects and shorten recovery times. However, when applied as a single injection (e.g., peripheral nerve block) it is limited by the duration of its effect. Herein, we develop a thermoresponsive nanogel based on poly(oligoethylene glycol methacrylate) containing the long-lasting anesthetic bupivacaine, which can be externally activated by using near-infrared light due to the photothermal properties of hollow gold nanoparticles embedded in the nanogel which facilitate its phase transition, triggering drug release at a controlled temperature above body temperature. Bupivacaine in vitro release can be repeatedly triggered to achieve a controlled pulsatile release of the drug due to the reversible nature of the thermosensitive nanogel, achieving a spatio-temporal control of the release. In vivo sciatic nerve block demonstrates that whereas the administered dose of free bupivacaine produces sensory block and impaired motor function for 2 h, the equivalent bupivacaine dose included in the developed release system can significantly prolong its neurobehavioral anesthetic effect for over 6 h. This release system can also be reactivated multiple times by subsequent irradiation cycles without observing detrimental toxicity in the infiltrated tissues., Financial support from the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM) is gratefully acknowledge. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund. We acknowledge the LMA-INA (University of Zaragoza, Spain), the Histopathology Unit from CNIO (Spain), and Cell Culture, Cell Separation and Flow Cytometry, Animal Care and Pathological Anatomy Core Units from IACS/IIS Aragon for their instruments and expertise. We would like to thank Dr. Elena Tapia, Dr. Jorge Palacio, Dr. Cristina Pastor and Dr. Eduardo Romanos for helpful advice in the in vivo studies. S.G-S. and G.L. acknowledge the support from the FPI program (BES-2015-073735 and PRE2018-085769, Spanish Ministry of Science, Innovation and Universities). G.M. gratefully acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III).

The management of skin and soft-tissue infections represents a burden for healthcare systems worldwide, demanding additional scientific efforts. Despite combined advances in modern medicine from different disciplines, chronic non-healing topical wounds still represent an unresolved clinical challenge. Nanotechnology has contributed significantly to the development of advanced therapeutic and diagnostic approaches in wound care. From this perspective, recommendations on the design of nano-based approaches for the management of infected non-healing chronic wounds are suggested. Preclinical results have demonstrated that nanostructured antimicrobial-loaded dressings and hydrogels can reduce the pathogenic bioburden and can restore the wound's physiological balance. Future clinical trials that ensure meaningful results are recommended., Financial support from the Spanish Ministry of Science and Innovation (Grant Nos. PID2020-113987RB-I00 and CTQ2017-84473-R) is gratefully acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund.

Synergistic antimicrobial effects were observed for copper sulfide (CuS) nanoparticles together with indocyanine green (ICG) in the elimination of wild type pathogenic bacteria (Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853) and also opportunistic fungal infective yeast (Candida albicans ATCC 10231). Furthermore, large antibacterial effects were observed for clinical isolates of Methicillin-resistant S. aureus (MRSA) PFGE strain-type USA300. This efficient antimicrobial action was attributed to the combined extra- and intracellular generation of reactive oxygen species upon light irradiation. Instead of the use of visible-light for the activation of common photosensitizers, both ICG and CuS nanoparticles can be activated in the near infrared (NIR)-region of the electromagnetic spectrum and therefore, superior tissue penetration would be expected in a potential elimination of pathogenic microorganisms not only on the skin but also in the soft tissue. In the different bacteria studied a 3-log reduction in the bacterial counts was achieved after only 6 min of NIR irradiation and treatment with ICG or CuS alone at concentrations of 40 and 160 µg mL−1, respectively. A maximum bactericidal effect against S. aureus and USA300 strains was obtained for the combination of both photosensitizers at the same concentration. Regarding P. aeruginosa, a 4-log reduction in the CFU was observed for the combination of CuS and ICG at various concentrations. In Candida albicans the combination of both ICG and CuS and light irradiation showed an antimicrobial dose-dependent effect with the reduction of at least 3-log in the cell counts for the combination of ICG + CuS at reduced concentrations. The observed antimicrobial effect was solely attributed to a photodynamic effect and any photothermal effect was avoided to discard any potential thermal injury in a potential clinical application. The generation of reactive oxygen species upon near infrared-light irradiation for those photosensitizers used was measured either alone or in combination. The cytocompatibility of the proposed materials at the doses used in photodynamic therapy was also demonstrated in human dermal fibroblasts and keratinocytes by cell culturing and flow cytometry studies., Financial support from Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00 is gratefully acknowledged. This research was also funded by the Spanish Ministry of Economy and Competitiveness (grant number CTQ2017-84473-R). 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 (Spain) with assistance from the European Regional Development Fund. We acknowledge the LMA-INA (University of Zaragoza, Spain). G. M. acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III).

By combining the photothermal ability of copper sulphide nanoparticles (NPs) upon excitation with Near Infrared (NIR) Light and the thermo-responsive properties of the homemade oligo (ethylene glycol) methyl ether methacrylate copolymer we have obtained fragmentable nanocomposites able to release a carried drug on-demand after NIR-light triggering. A complete physico-chemical characterization of the resulting nanoparticles has been carried out and their degradation assessed at different temperatures. Herein, we have also evaluated the drug loading capacity of those nanoparticles and the temperature dependence in their drug release kinetics using bupivacaine hydrochloride as a model drug. For those hybrid nanoparticles, subcytotoxic doses on four different cell lines and their potential interference in cell metabolism, induction of apoptosis, and cell cycle have been evaluated by Alamar Blue fluorometry and flow cytometry., The financial support was provided by the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM), which is gratefully acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund.

Tailoring surface properties by layer-by-layer (LBL) deposition directed on the construction of complex multilayer coatings with nanoscale precision enables the development of novel structures and devices with desired functional properties (i.e., osseointegration, bactericidal activity, biocorrosion protection). Herein, electrostatic self-assembly was applied to fabricate biopolymer-based coatings involving chitosan (CSM) and alginate (AL) enriched with caffeic acid (CA) on Ti-6Al-7Nb alloyed surfaces. The method of CA grafting onto the chitosan backbone (CA-g-CSM) as well as all used reagents for implant functionalization were chosen as green and sustainable approach. The final procedure of surface modification of the Ti-6Al-7Nb alloy consists of three steps: (i) chemical treatment in Piranha solution, (ii) plasma chemical-activation of the Ti alloy surface in a RF CVD (Radio Frequency Chemical Vapour Deposition) reactor using Ar, O2 and NH3 gaseous precursors, and (iii) a multi-step deposition of bio-functional coatings via dip-coating method. Corrosion tests have revealed that the resulting chitosan-based coatings, also these involving CA, block the specimen surface and hinder corrosion of titanium alloy. Furthermore, the antioxidant layers are characterized by beneficial level of roughness (Ra up ca. 350 nm) and moderate hydrophilicity (59°) with the dispersion part of conducive surface energy ca. 30 mJ/m2. Noteworthy, all coatings are biocompatible as the intact morphology of cultured eukaryotic cells ensured proper growth and proliferation, while exhibit bacteriostatic character, particularly in contact with Gram-(−) bacteria (E. coli). The study indicates that the applied simple sustainable strategy has contributed significantly to obtaining homogeneous, stable, and biocompatible while antibacterial biopolymer-based coatings., This work has been supported by the National Science Centre (NCN) (grant decision no. DEC-2017/01/X/ST8/00886).

Bovine Serum Albumin (BSA) and Horseradish Peroxidase (HRP) have been encapsulated within microparticulated matrices composed of Eudragit RS100 by the water-in-oil-in-water double emulsion solvent evaporation method. Good encapsulation efficiencies were achieved for BSA and HRP, 88.4 and 95.8%, respectively. The stability of the loaded proteins was confirmed by using circular dichroism and fluorescence. The gastroresistance of the protein-loaded microparticles was evaluated under simulated gastric conditions demonstrating the preservation of the structural integrity of the proteins loaded inside the particles. The enzymatic activity of HRP after being released from the enteric microparticles was evaluated by using the peroxidase substrate, revealing that the released enzyme preserved its 100% function. The high drug loadings achieved, reduced cytotoxicity and efficient gastric protection point out towards the potential use of those carriers as oral delivery vectors of therapeutic proteins offering a more controlled targeted release in specific sites of the intestine and an enhanced gastrointestinal absorption.

The development of thermoresponsive nanogels loaded with nanocrystals of the local anesthetic bupivacaine nanocrystals (BNCs) for prolonged peripheral nerve pain relief is reported here. BNCs were prepared using the antisolvent precipitation method from the hydrophobic form of bupivacaine (bupivacaine free base). The as-prepared BNCs were used stand-alone or encapsulated in temperature-responsive poly(ethylene glycol) methyl ether methacrylate (OEGMA)-based nanogels, resulting in bupivacaine NC-loaded nanogels (BNC-nanogels) of monodisperse size. The synthesis protocol has rendered high drug loadings (i.e., 93.8 ± 1.5 and 84.8 ± 1.2 wt % for the NC and BNC-nanogels, respectively) and fast drug dissolution kinetics in the resulting composite material. In vivo tests demonstrated the efficacy of the formulation along with an extended duration of sciatic nerve block in murine models of more than 8 h with a formulation containing only 2 mg of the local anesthetic thanks to the thermoresponsive character of the polymer, which, at body temperature, becomes hydrophobic and acts as a diffusion barrier for the encapsulated drug nanocrystals. The hydrophobicity of the encapsulated bupivacaine free base probably facilitates its pass through cell membranes and also binds strongly to their hydrophobic lipid bilayer, thereby protecting molecules from diffusion to extracellular media and to the bloodstream, reducing their clearance. When using BNC-nanogels, the duration of the anesthetic blockage lasted twice as long as compared to the effect of just BNCs or a conventional bupivacaine hydrochloride solution both containing equivalent amounts of the free drug. Results of the in vivo tests showed enough sensory nerve block to potentially relieve pain, but still having mobility in the limb, which enables motor function when required. The BNC-nanogels presented minimal toxicity in the in vivo study due to their sustained drug release and excellent biocompatibility. The encapsulation of nano-sized crystals of bupivacaine provides a prolonged regional anesthesia with reduced toxicity, which could be advantageous in the management of chronic pain., The authors thank financial support from the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM). V.S. acknowledges the financial support from Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund. The microscopy works have been conducted in the “Laboratorio de Microscopias Avanzadas” at “Instituto de Nanociencia de Aragon─Universidad de Zaragoza”. The authors acknowledge the LMA-INA, the Histopathology Unit from CNIO (Madrid, Spain), and Cell Separation and Flow Cytometry, Cell Culture, Animal Care and Pathological Anatomy Core Units from IACS/IIS Aragon (Spain) for granting access to their instruments and expertise. The authors also acknowledge Drs. Elena Tapia, Jorge Palacio, Cristina Pastor, and Eduardo Romanos for their helpful advice and comments regarding the in vivo model. S.G.-S. and G.L. gratefully acknowledge the support from the FPI program (BES-2015-073735 and PRE2018-085769). G.M. thanks the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III).

In resource-limited settings, fast and simple point-of-need tests should facilitate healthcare providers the identification of pathogens avoiding empirical suboptimal treatments with broad-spectrum antibiotics. A rapid optical whole cell bacterial biosensor has been here developed using sialic acid functionalized gold nanoparticles allowing the selective screening of Gram-positive Staphylococcus aureus ATCC 25923 and Methicillin Resistant Staphylococcus aureus (MRSA) USA300 and Gram-negative bacteria (Pseudomonas aeruginosa ATCC 15442) by selecting the appropriate dispersing media. Those bacteria were selected due to their common presence in wound bed tissue of chronic infected topical wounds. The discrimination of bacterial pathogens has been attempted in different media including water, two independent buffers, bacterial broth, human serum and human urine. The identification of Gram + bacterial pathogens was also assessed under simultaneous co-culture of S. Aureus and Pseudomonas aeruginosa. High bacterial loads were required to provide with a statistically significant optical pathogen identification in human serum whereas it was not possible to detect the presence of bacteria at clinically relevant levels in urine., This research was funded by the Spanish Ministry of Economy and Competitiveness (grant number CTQ2014-52384-R). Financial support from the EU thanks to the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM) is also acknowledged. G.L. acknowledges the support from the FPI fellowship PRE2018-085769 granted by the Spanish Ministry of Science, Innovation and Universities, while L.G.M-C. acknowledges the financial support from the Mexican Council of Science and Technology (CONACyT) through doctoral grant#710618. G.M. gratefully acknowledges the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III). VS acknowledges the LMA-INA for offering access to their instruments and the financial support of Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00. 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 (Spain) with assistance from the European Regional Development Fund.

Purpose: The search of novel molecules for the treatment of osteoarthritis (OA) is complex as any new therapeutic approach should encompass these requirements: inhibition of cartilage degradation, protection of bone and inhibition of inflammation. In the last years, different drugs have been proposed though most of them did not succeed in fulfil these requirements. Moreover, few of them have been encapsulated in drug delivery systems to improve their therapeutic potential to achieve a sustained or controlled release compared to the administration of equivalent doses of the free compounds. Nanoscience has arisen in the last decades as a potential field of study in drug delivery because nanomaterials may overcome the main current limitations to achieve an efficient and localized drug delivery by improving the targeted delivery and providing a sustained or controlled delivery to prolong the therapeutic effect. On the other hand, different polyphenols and aromatic organic compounds are known to possess anti-inflammatory, antioxidant and bone density-building properties...

This article belongs to the Special Issue Functional Polymeric Nanoparticles., The development of new gene-editing technologies has fostered the need for efficient and safe vectors capable of encapsulating large nucleic acids. In this work we evaluate the synthesis of large-size plasmid-loaded PLGA nanoparticles by double emulsion (considering batch ultrasound and microfluidics-assisted methodologies) and magnetic stirring-based nanoprecipitation synthesis methods. For this purpose, we characterized the nanoparticles and compared the results between the different synthesis processes in terms of encapsulation efficiency, morphology, particle size, polydispersity, zeta potential and structural integrity of loaded pDNA. Our results demonstrate particular sensibility of large pDNA for shear and mechanical stress degradation during double emulsion, the nanoprecipitation method being the only one that preserved plasmid integrity. However, plasmid-loaded PLGA nanoparticles synthesized by nanoprecipitation did not show cell expression in vitro, possibly due to the slow release profile observed in our experimental conditions. Strong electrostatic interactions between the large plasmid and the cationic PLGA used for this synthesis may underlie this release kinetics. Overall, none of the methods evaluated satisfied all the requirements for an efficient non-viral vector when applied to large-size plasmid encapsulation. Further optimization or alternative synthesis methods are thus in current need to adapt PLGA nanoparticles as delivery vectors for gene editing therapeutic technologies., This research was supported by the Instituto de Salud Carlos III, grant number PI17/00949; the Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” from the European Union; the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED612-CB18/05/00037); the Horizon 2020 framework Programme Marie Curie individual fellowship scheme to R.M. (Fellowship 752349); and Consolidated Groups from Gobierno de Aragón. V.S. acknowledges the LMA-INA for offering access to their instruments and the financial support of Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyect REF: RTI2018-099019-A-I00. 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 (Spain) with assistance from the European Regional Development Fund.

This article belongs to the Special Issue State-of-the-Art Polymer Science and Technology in Spain (2020,2021)., Hybrid linear–dendritic block copolymers (LDBCs) having dendrons with a precise number of peripheral groups that are able to supramolecular bind functional moieties are challenging materials as versatile polymeric platforms for the preparation of functional polymeric nanocarriers. PEG2k-b-dxDAP LDBCs that are based on polyethylene glycol (PEG) as hydrophilic blocks and dendrons derived from bis-MPA having 2,6-diacylaminopyridine (DAP) units have been efficiently synthesized by the click coupling of preformed blocks, as was demonstrated by spectroscopic techniques and mass spectrometry. Self-assembly ability was first checked by nanoprecipitation. A reproducible and fast synthesis of aggregates was accomplished by microfluidics optimizing the total flow rate and phase ratio to achieve spherical micelles and/or vesicles depending on dendron generation and experimental parameters. The morphology and size of the self-assemblies were studied by TEM, Cryogenic Transmission Electron Microscopy (cryo-TEM), and Dynamic Light Scattering (DLS). The cytotoxicity of aggregates synthesized by microfluidics and the influence on apoptosis and cell cycle evaluation was studied on four cell lines. The self-assemblies are not cytotoxic at doses below 0.4 mg mL−1. Supramolecular functionalization using thymine derivatives was explored for reversibly cross-linking the hydrophobic blocks. The results open new possibilities for their use as drug nanocarriers with a dynamic cross-linking to improve nanocarrier stability but without hindering disassembly to release molecular cargoes., This work was supported by the Ministerio de Economía y Competitividad (MINECO)-FEDER, Spain, under the project grant number MAT107-84838-P (L.O., M.P., Programa Excelencia), Fondo Social Europeo and Gobierno de Aragon-FEDER (E47_17R, FEDER 2014-2020 “Construyendo Europa desde Aragón”). M. Abad (M.Ab.) acknowledges Gobierno de Aragón and Fondo Social Europeo for her PhD grant. G. Mendoza (G.M.) is also grateful for the support from the Miguel Servet Program (MS19/00092; Instituto de Salud Carlos III). Authors acknowledge the Centro de Química y Materiales de Aragón (CEQMA) for the NMR and Thermal Analysis facilities, and the use of Electron Microscopy facilities of the Laboratorio de Microscopías Avanzadas (LMA). V. Sebastián (V.S.) acknowledges the financial support of Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00.

The incorporation of plasmonic nanoconstructs in biodegradable polymeric nanoparticles (NPs), together with therapeutic drugs in a controlled procedure is of interest for different applications in Nanomedicine. Advanced hybrid nanomaterials can be engineered by combining the in situ formation of plasmonic palladium nanosheets (NSs) and the proper ionic nature of the encapsulated drug. This study presents a new procedure to synthesize hybrid nanostructures by a Pickering double emulsion. Anisotropic palladium (Pd) NSs with unique near-infrared (NIR)-optical properties can be assembled within a poly lactic-co-glycolic acid matrix of < 200 nm NPs, when Pd precursors are in situ reduced via a gas-phase procedure. The hybrid nanomaterials respond to external NIR light stimulus. The unprecedented precision to assemble, in a single stage, plasmonic nanoconstructs having a total loading selectivity, when encapsulated in combination with hydrophobic drugs, offers new opportunities in the new class of theragnostics, in particular when triggered drug delivery and photothermal therapies are required., The authors thank financial support from the ERC Consolidator Grant program (No. ERC-2013-CoG-614715, NANOHEDONISM). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund. The (HR)TEM and STEM-EDS studies were conducted at the Laboratorio de Microscopias Avanzadas, Universidad de Zaragoza, Spain. G.M. thanks the support from the Miguel Servet Program (No. MS19/00092; Instituto de Salud Carlos III). V.S. acknowledges the financial support of Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto >REF: RTI2018-099019-A-I00. R.A. acknowledges funding from the Spanish MICINN (project grant PID2019-104739GB-100/AEI/10.13039/501100011033), from the Government of Aragon (project DGA E13-20R), and from the European Union H2020 program “ESTEEM3” (No. 823717).

The use of exosomes as selective delivery vehicles of therapeutic agents, such as drugs or hyperthermia-capable nanoparticles, is being intensely investigated on account of their preferential tropism toward their parental cells. However, the methods used to introduce a therapeutic load inside exosomes often involve disruption of their membrane, which may jeopardize their targeting capabilities, attributed to their surface integrins. On the other hand, in recent years bio-orthogonal catalysis has emerged as a new tool with a myriad of potential applications in medicine. These bio-orthogonal processes, often based on Pd-catalyzed chemistry, would benefit from systems capable of delivering the catalyst to target cells. It is therefore highly attractive to combine the targeting capabilities of exosomes and the bio-orthogonal potential of Pd nanoparticles to create new therapeutic vectors. In this protocol, we provide detailed information on an efficient procedure to achieve a high load of catalytically active Pd nanosheets inside exosomes, without disrupting their membranes. The protocol involves a multistage process in which exosomes are first harvested, subjected to impregnation with a Pd salt precursor followed by a mild reduction process using gas-phase CO, which acts as both a reducing and growth-directing agent to produce the desired nanosheets. The technology is scalable, and the protocol can be conducted by any researcher having basic biology and chemistry skills in ~3 d., We gratefully acknowledge financial support from the ERC Advanced Grant CADENCE (grant no. ERC-2016-ADG-742684) and the EPSRC (Healthcare Technology Challenge award no. EP/N021134/1). M.S.-A. thanks the Spanish Government for an FPU PhD research fellowship. B.R.-R. thanks the EC (grant no. H2020-MSCA-IF-2014–658833). V.S. acknowledges the financial support of Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00. M.A. acknowledges the financial support of the ERC Consolidator Grant programme (grant no. ERC-2013-CoG-614715). P.M.-D. also thanks Instituto de Salud Carlos III (PI19/01007). We also thank CIBER-BBN, an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III and by Fondo Europeo de Desarrollo Regional (Feder) ‘Una manera de hacer Europa’, with the assistance of the European Regional Development Fund. This study is also partially funded by the Aragon Government (T57_17R p) cofounded by Feder 2014–2020 ‘Building Europe from Aragon’.