PLGA nanoparticle-encapsulated lysostaphin for the treatment of Staphylococcus aureus infections.

Staphylococcus aureus possesses the ability to become pathogenic, leading to severe and life-threatening infections. Its methicillin-resistant variant MRSA has garnered high-priority status due to its increased morbidity and associated mortality. This emphasizes the urgency for novel anti-staphylococcal agents. The bacteriocin lysostaphin stands out for its remarkable bactericidal activity against S. aureus, including MRSA, outperforming conventional antibiotics. However, the clinical application of lysostaphin faces challenges, including enzymatic activity loss under physiological conditions and potential immunogenicity. This study introduces a novel approach by encapsulating lysostaphin within polylactic-co-glycolic acid (PLGA) nanoparticles, a biodegradable copolymer known for its biocompatibility and sustained drug release ability. The study assesses the antimicrobial activity of lysostaphin-loaded PLGA nanoparticles against different S. aureus strains, and we also used GFP-expressing S. aureus for facilitating its traceability in planktonic, biofilm, and intracellular infection models. The results showed the significant reduction in bacteria viability both in planktonic and biofilm states. The in vitro intracellular infection model demonstrated the significantly enhanced efficiency of the developed nanoparticles compared to the treatment with the free bacteriocin. This research presents lysostaphin encapsulation within PLGA nanoparticles and offers promising avenues for enhancing lysostaphin's therapeutic efficacy against S. aureus infections.

Antimicrobial Photodynamic Therapy Using Encapsulated Protoporphyrin IX for the Treatment of Bacterial Pathogens.

Herein, we report on the antimicrobial photodynamic effect of polymeric nanoparticles containing the endogenous photosensitizer protoporphyrin IX. Compared to equivalent doses of the free photosensitizer, we demonstrated that the photodynamic antimicrobial efficacy of PLGA (polylactic-co-glycolic acid) nanoparticles containing protoporphyrin IX (PpIX) against pathogenic Staphylococcus aureus (S. aureus) is preserved after encapsulation, while photobleaching is reduced. In addition, compared to equivalent doses of the free porphyrin, we show that a reduction in the cytotoxicity in mammalian cell cultures is observed when encapsulated. Therefore, the encapsulation of protoporphyrin IX reduces its photodegradation, while the released photosensitizer maintains its ability to generate reactive oxygen species upon light irradiation. The polymeric nanoencapsulation promotes aqueous solubility for the hydrophobic PpIX, improves its photostability and reduces the cytotoxicity, while providing an extended release of this endogenous photosensitizer.

Gold Nanoparticles Capped with a Novel Titanium(IV)-Containing Polyoxomolybdate Cluster: Selective and Enhanced Bactericidal Effect Against Escherichia coli.

Bacterial infections are a public health threat of increasing concern in medical care systems; hence, the search for novel strategies to lower the use of antibiotics and their harmful effects becomes imperative. Herein, the antimicrobial performance of four polyoxometalate (POM)-stabilized gold nanoparticles (Au@POM) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as Gram-negative and Gram-positive bacteria models, respectively, is studied. The bactericidal studies performed, both in planktonic and sessile forms, evidence the antimicrobial potential of these hybrid nanostructures with selectivity toward Gram-negative species. In particular, the Au@GeMoTi composite with the novel [Ti2 (HGeMo7 O28 )2 ]10- POM capping ligand exhibits outstanding bactericidal efficiency with a minimum inhibitory concentration of just 3.12 µm for the E. coli strain, thus outperforming the other three Au@POM counterparts. GeMoTi represents the fourth example of a water-soluble TiIV -containing polyoxomolybdate, and among them, the first sandwich-type structure having heteroatoms in high-oxidation state. The evaluation of the bactericidal mechanisms of action points to the cell membrane hyperpolarization, disruption, and subsequent nucleotide leakage and the low cytotoxicity exerted on five different cell lines at antimicrobial doses demonstrates the antibiotic-like character. These studies highlight the successful design and development of a new POM-based nanomaterial able to eradicate Gram-negative bacteria without damaging mammalian cells.

Real-time in vivo monitoring of the antimicrobial action of combination therapies in the management of infected topical wounds.

The increasing prevalence of non-healing infected wounds has become a serious concern in the clinical practice, being associated to population aging and to the rising prevalence of several chronic conditions such as diabetes. Herein, the evaluation of the bactericidal and antibiofilm effects of the natural antiseptic terpenes thymol and farnesol standing alone or in combination with the standard care antiseptic chlorhexidine was carried out both in vitro and in vivo. The in vitro combinatorial treatment of chlorhexidine associated with those terpenes against Staphylococcus aureus in its planktonic and sessile forms demonstrated a superior antibacterial activity than that of chlorhexidine alone. Real-time in vivo monitoring of infection progression and antimicrobial treatment outcomes were evaluated using the bioluminescent S. aureus strain Xen36. In vivo studies on infected wound splinting murine models corroborated the superior bactericidal effects of the combinatorial treatments here proposed. Moreover, the encapsulation of thymol in electrospun Eudragit® S100 (i.e., a synthetic anionic copolymer of methacrylic acid and ethyl acrylate)-based wound dressings was also carried out in order to design efficient antimicrobial wound dressings.

Antibody-Functionalized Polymer Nanoparticles for Targeted Antibiotic Delivery in Models of Pathogenic Bacteria Infecting Human Macrophages.

The efficacy of antibody-functionalized poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs), prepared by nanoprecipitation, carrying rifampicin (RIF) against planktonic, sessile, and intracellular Staphylococcus aureus and Escherichia coli is reported here. A biotinylated anti-S. aureus polyclonal antibody, which binds to structural antigens of the whole bacterium, was functionalized on the surface of RIF-loaded PLGA-based NPs by using the high-affinity avidin-biotin complex. This general strategy allows the binding of commercially available biotinylated antibodies. Coculture models of S. aureus ATCC 25923 and Escherichia coli S17 were used to demonstrate the preferential selectivity of the antibody-functionalized NPs against the Gram-positive bacterium only. At 0.2 µg/mL, complete S. aureus eradication was observed for the antibody-functionalized RIF-loaded NPs, whereas only a 5-log reduction was observed for the nontargeted RIF-loaded NPs. S. aureus is a commensal facultative pathogen having part of its live cycle intracellularly in both phagocytic and nonphagocytic cells. Those intracellular bacterial persisters, named small colony variants, have been postulated as reservoirs of relapsed episodes of infection and consequent treatment failure. At 0.5 µg/mL, the RIF-loaded NPs reduced in 2-log intracellular S. aureus-infecting human macrophages. The ability of those antibody-functionalized nanoparticles to prevent biofilm formation or to reduce the bacterial burden in already-formed mature biofilms is also reported here using S. aureus and E. coli single and cocultured biofilms. In the prevention of S. aureus biofilm formation, the antibody-functionalized NPs exerted a superior inhibition of bacterial growth (up to 2 logs) compared to the nonfunctionalized ones. This study demonstrates the selectivity of the synthesized immunonanoparticles and their antimicrobial efficacy in different scenarios, including planktonic cultures, sessile conditions, and even against intracellular infective pathogens.

The E1a Adenoviral Gene Upregulates the Yamanaka Factors to Induce Partial Cellular Reprogramming.

The induction of pluripotency by enforced expression of different sets of genes in somatic cells has been achieved with reprogramming technologies first described by Yamanaka's group. Methodologies for generating induced pluripotent stem cells are as varied as the combinations of genes used. It has previously been reported that the adenoviral E1a gene can induce the expression of two of the Yamanaka factors (c-Myc and Oct-4) and epigenetic changes. Here, we demonstrate that the E1a-12S over-expression is sufficient to induce pluripotent-like characteristics closely to epiblast stem cells in mouse embryonic fibroblasts through the activation of the pluripotency gene regulatory network. These findings provide not only empirical evidence that the expression of one single factor is sufficient for partial reprogramming but also a potential mechanistic explanation for how viral infection could lead to neoplasia if they are surrounded by the appropriate environment or the right medium, as happens with the tumorogenic niche.

Elucidating the mechanisms of action of antibiotic-like ionic gold and biogenic gold nanoparticles against bacteria.

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.

New insights in osteoarthritis diagnosis and treatment: Nano-strategies for an improved disease management.

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 article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement.

Anti-Inflammatory and Chondroprotective Effects Induced by Phenolic Compounds from Onion Waste Extracts in ATDC-5 Chondrogenic Cell Line.

Osteoarthritis is a prevalent degenerative condition that is closely related to the destruction and inflammation of cartilage. The high prevalence of this pathology exhorts researchers to search for novel therapeutic approaches. Vegetable-fruit wastes have emerged as a promising origin of anti-inflammatory and antioxidant compounds that, in some cases, may also exert chondroprotective effects. This study aims to decipher the potential of onion waste products in the inhibition of molecular events involved in osteoarthritis. Onion extracts showed a high content of phenolic compounds and antioxidant properties. Cytocompatibility was demonstrated in the chondrogenic cell line ATDC-5, exerting viability percentages higher than 90% and a slight increase in the S phase cycle cell. The induction of inflammation mediated by the lipopolysaccharide and onion extracts' treatment substantially inhibited molecular markers related to inflammation and cartilage degradation, highlighting the promising application of onion extracts in biomedical approaches. The in silico analyses suggested that the results could be attributed to protocatechuic, ellagic, and vanillic acids' greater cell membrane permeability. Our work provides distinctive information about the possible application of waste onion extracts as functional components with anti-inflammatory and chondroprotective characteristics in osteoarthritis.

Pharmacokinetic control on the release of antimicrobial drugs from pH-responsive electrospun wound dressings.

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.

Valorization of Onion Waste by Obtaining Extracts Rich in Phenolic Compounds and Feasibility of Its Therapeutic Use on Colon Cancer.

In this study, the total phenolic content, the antioxidant and antiproliferative activities of onion waste extracts were characterized. Some phenolic compounds present in the extracts were also identified and quantified by HPLC-DAD. Additionally, an in-silico analysis was performed to identify the phenolic compounds with the highest intestinal absorption and Caco-2 permeability. The onion extract possessed a high amount of phenolic compounds (177 ± 9 mg/g extract) and had an effective antioxidant capacity measured by ABTS, FRAP and DPPH assays. Regarding the antiproliferative activity, the onion extracts produced cell cycle arrest in the S phase with p53 activation, intrinsic apoptosis (mitochondrial membrane potential modification) and caspase 3 activation. Likewise, onion waste increased intracellular ROS with possible NF-kB activation causing a proteasome down regulation. In addition, the extracts protected the intestine against oxidative stress induced by H2O2. According to the in-silico analysis, these results could be related to the higher Caco-2 permeability to protocatechuic acid. Therefore, this study provides new insights regarding the potential use of these types of extract as functional ingredients with antioxidant and antiproliferative properties and as medicinal agents in diseases related to oxidative stress, such as cancer. In addition, its valorization would contribute to the circular economy.

Light activated pulsatile drug delivery for prolonged peripheral nerve block.

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.

Microfluidic Synthesis of Block Copolymer Micelles: Application as Drug Nanocarriers and as Photothermal Transductors When Loading Pd Nanosheets.

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.

Hybrid thermoresponsive nanoparticles containing drug nanocrystals for NIR-triggered remote release.

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.

Selective point-of-care detection of pathogenic bacteria using sialic acid functionalized gold nanoparticles.

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.

Chalcogenide nanoparticles and organic photosensitizers for synergetic antimicrobial photodynamic therapy.

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.

Nanogels with High Loading of Anesthetic Nanocrystals for Extended Duration of Sciatic Nerve Block.

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.

Supramolecular Functionalizable Linear-Dendritic Block Copolymers for the Preparation of Nanocarriers by Microfluidics.

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.

Antimicrobial Wound Dressings against Fluorescent and Methicillin-Sensitive Intracellular Pathogenic Bacteria.

There is limited evidence indicating that drug-eluting dressings are clinically more effective than simple conventional dressings. To shed light on this concern, we have performed evidence-based research to evaluate the antimicrobial action of thymol (THY)-loaded antimicrobial dressings having antibiofilm forming ability, able to eradicate intracellular and extracellular pathogenic bacteria. We have used four different Staphylococcus aureus strains, including the ATCC 25923 strain, the Newman strain (methicillin-sensitive strain, MSSA) expressing the coral green fluorescent protein from the vector pCN47, and two clinical reference strains, Newman-(MSSA) and USA300-(methicillin-resistant strain), as traceable models of pathogenic bacteria commonly infecting skin and soft tissues. Compared to non-loaded dressings, THY-loaded polycaprolactone-based electrospun dressings were also able to eliminate pathogenic bacteria in coculture models based on infected murine macrophages. In addition, by using confocal microscopy and the conventional microdilution plating method, we corroborated the successful ability of THY in preventing also biofilm formation. Herein, we demonstrated that the use of wound dressings loaded with the natural monoterpenoid phenol derivative THY are able to eliminate biofilm formation and intracellular methicillin-sensitive S aureus more efficiently than with their corresponding THY-free counterparts.

Isolation of exosomes from whole blood by a new microfluidic device: proof of concept application in the diagnosis and monitoring of pancreatic cancer.

BACKGROUND: Exosomes are endocytic-extracellular vesicles with a diameter around 100 nm that play an essential role on the communication between cells. In fact, they have been proposed as candidates for the diagnosis and the monitoring of different pathologies (such as Parkinson, Alzheimer, diabetes, cardiac damage, infection diseases or cancer). RESULTS: In this study, magnetic nanoparticles (Fe3O4NPs) were successfully functionalized with an exosome-binding antibody (anti-CD9) to mediate the magnetic capture in a microdevice. This was carried out under flow in a 1.6 mm (outer diameter) microchannel whose wall was in contact with a set of NdFeB permanent magnets, giving a high magnetic field across the channel diameter that allowed exosome separation with a high yield. To show the usefulness of the method, the direct capture of exosomes from whole blood of patients with pancreatic cancer (PC) was performed, as a proof of concept. The captured exosomes were then subjected to analysis of CA19-9, a protein often used to monitor PC patients. CONCLUSIONS: Here, we describe a new microfluidic device and the procedure for the isolation of exosomes from whole blood, without any need of previous isolation steps, thereby facilitating translation to the clinic. The results show that, for the cases analyzed, the evaluation of CA19-9 in exosomes was highly sensitive, compared to serum samples.