Melanin-Based Nanoparticles for Lymph Node Tattooing: Experimental, Histopathological and Ultrastructural Study.

In breast cancer, Targeted Axillary Dissection (TAD) allows for the selective excision of the sentinel lymph node (SLN) during primary tumor surgery. TAD consists of the resection of labelled SLNs prior to neoadjuvant chemotherapy (NACT). Numerous clinical and preclinical studies have explored the use of carbon-based colloids for SLN tattooing prior to NACT. However, carbon vectors show varying degrees of inflammatory reactions and, in about one fifth of cases, carbon particles migrate via the lymphatic pathway to other nodes, causing the SLN to mismatch the tattooed node. To overcome these limitations, in this study, we explored the use of melanin as a staining endogenous pigment. We synthesized and characterized melanin-loaded polymeric nanoparticles (Mel-NPs) and used them to tattoo lymph nodes in pig animal models given the similarity in the size of the human and pig nodes. Mel-NPs tattooed lymph nodes showed high identification rates, reaching 83.3% positive identification 16 weeks after tattooing. We did not observe any reduction in the identification as time increased, implying that the colloid is stable in the lymph node tissue. In addition, we performed histological and ultrastructural studies to characterize the biological behavior of the tag. We observed foreign-body-like granulomatous inflammatory responses associated with Mel-NPs, characterized by the formation of multinucleated giant cells. In addition, electron microscopy studies showed that uptake is mainly performed by macrophages, and that macrophages undergo cellular damage associated with particle uptake.

Expert opinion on antimicrobial therapies: is there enough scientific evidence to state that targeted therapies outperform non-targeted ones?

INTRODUCTION: Different active and passive strategies have been developed to fight against pathogenic bacteria. Those actions are undertaken to reduce the bacterial burden while minimizing the possibilities to develop not only antimicrobial resistance but also antimicrobial side-effects such as allergic or hypersensitivity reactions. AREAS COVERED: We have reviewed preclinical results that evidence that targeted antimicrobial therapies outperform non-targeted ones. Active selective targeting against pathogenic bacteria has been achieved through the functionalization of antimicrobials, either alone or encapsulated within micro- or nanocarriers, with various recognition moieties. These moieties include peptides, aptamers, antibodies, carbohydrates, extracellular vesicles, cell membranes, infective agents, and other affinity ligands with specific bacterial tropism. Those selective ligands increase retention and enhance effectiveness reducing the side-effects and the required dose to exert the antimicrobial action at the site of infection. EXPERT OPINION: When using targeted antimicrobial therapies not only reduced side-effects are observed, but also, compared to the administration of equivalent doses of the non-targeted drugs, a superior efficacy has been demonstrated against planktonic, sessile, and intracellular pathogenic bacterial persisters. The translation of those targeted therapies to subsequent phases of clinical development still requires the demonstration of a reduction in the probabilities for the pathogen to develop resistance when using targeted approaches.

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.

Encapsulation of Iron-Saturated Lactoferrin for Proteolysis Protection with Preserving Iron Coordination and Sustained Release.

Lactoferrin (Lf) is a globular glycoprotein found mainly in milk. It has a very high affinity for iron(III) ions, and its fully saturated form is called holoLf. The antimicrobial, antiviral, anticancer, and immunomodulatory properties of Lf have been studied extensively for the past two decades. However, to demonstrate therapeutic benefits, Lf has to be efficiently delivered to the intestinal tract in its structurally intact form. This work aimed to optimize the encapsulation of holoLf in a system based on the versatile Eudragit® RS polymer to protect Lf against the proteolytic environment of the stomach. Microparticles (MPs) with entrapped holoLf were obtained with satisfactory entrapment efficiency (90-95%), high loading capacity (9.7%), and suitable morphology (spherical without cracks or pores). Detailed studies of the Lf release from the MPs under conditions that included simulated gastric or intestinal fluids, prepared according to the 10th edition of the European Pharmacopeia, showed that MPs partially protected holoLf against enzymatic digestion and ionic iron release. The preincubation of MPs loaded with holoLf under conditions simulating the stomach environment resulted in the release of 40% of Lf from the MPs. The protein released was saturated with iron ions at 33%, was structurally intact, and its iron scavenging properties were preserved.

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.

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.

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.

Submicronic Filtering Media Based on Electrospun Recycled PET Nanofibers: Development, Characterization, and Method to Manufacture Surgical Masks.

The disposal of single-use personal protective equipment has brought a notable environmental impact in the context of the COVID-19 pandemic. During these last two years, part of the global research efforts has been focused on preventing contagion using nanotechnology. This work explores the production of filter materials with electrohydrodynamic techniques using recycled polyethylene terephthalate (PET). PET was chosen because it is one of the materials most commonly present in everyday waste (such as in food packaging, bags, or bottles), being the most frequently used thermoplastic polymer in the world. The influence of the electrospinning parameters on the filtering capacity of the resulting fabric was analyzed against both aerosolized submicron particles and microparticulated matter. Finally, we present a new scalable and straightforward method for manufacturing surgical masks by electrospinning and we validate their performance by simulating the standard conditions to which they are subjected to during use. The masks were successfully reprocessed to ensure that the proposed method is able to reduce the environmental impact of disposable face masks.

Encapsulation of Large-Size Plasmids in PLGA Nanoparticles for Gene Editing: Comparison of Three Different Synthesis Methods.

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.

Controlling Particle Size and Release Kinetics in the Sustained Delivery of Oral Antibiotics Using pH-Independent Mucoadhesive Polymers.

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.

Microflow Nanoprecipitation of Positively Charged Gastroresistant Polymer Nanoparticles of Eudragit® RS100: A Study of Fluid Dynamics and Chemical Parameters.

The objective of the present work was to produce gastroresistant Eudragit® RS100 nanoparticles by a reproducible synthesis approach that ensured mono-disperse nanoparticles under the size of 100 nm. Batch and micromixing nanoprecipitation approaches were selected to produce the demanded nanoparticles, identifying the critical parameters affecting the synthesis process. To shed some light on the formulation of the targeted nanoparticles, the effects of particle size and homogeneity of fluid dynamics, and physicochemical parameters such as polymer concentration, type of solvent, ratio of solvent to antisolvent, and total flow rate were studied. The physicochemical characteristics of resulting nanoparticles were studied applying dynamic light scattering (DLS) particle size analysis and electron microscopy imaging. Nanoparticles produced using a micromixer demonstrated a narrower and more homogenous distribution than the ones obtained under similar conditions in conventional batch reactors. Besides, fluid dynamics ensured that the best mixing conditions were achieved at the highest flow rate. It was concluded that nucleation and growth events must also be considered to avoid uncontrolled nanoparticle growth and evolution at the collection vial. Further, rifampicin-encapsulated nanoparticles were prepared using both approaches, demonstrating that the micromixing-assisted approach provided an excellent control of the particle size and polydispersity index. Not only the micromixing-assisted nanoprecipitation promoted a remarkable control in the nanoparticle formulation, but also it enhanced drug encapsulation efficiency and loading, as well as productivity. To the best of our knowledge, this was the very first time that drug-loaded Eudragit® RS100 nanoparticles (NPs) were produced in a continuous fashion under 100 nm (16.5 ± 4.3 nm) using microreactor technology. Furthermore, we performed a detailed analysis of the influence of various fluid dynamics and physicochemical parameters on the size and uniformity of the resulting nanoparticles. According to these findings, the proposed methodology can be a useful approach to synthesize a myriad of nanoparticles of alternative polymers.

The Effect of Titanium Dioxide Surface Modification on the Dispersion, Morphology, and Mechanical Properties of Recycled PP/PET/TiO2 PBNANOs.

Titanium dioxide (TiO2) nanoparticles have recently appeared in PET waste because of the introduction of opaque PET bottles. We prepare polymer blend nanocomposites (PBNANOs) by adding hydrophilic (hphi), hydrophobic (hpho), and hydrophobically modified (hphoM) titanium dioxide (TiO2) nanoparticles to 80rPP/20rPET recycled blends. Contact angle measurements show that the degree of hydrophilicity of TiO2 decreases in the order hphi > hpho > hphoM. A reduction of rPET droplet size occurs with the addition of TiO2 nanoparticles. The hydrophilic/hydrophobic balance controls the nanoparticles location. Transmission electron microscopy (TEM_ shows that hphi TiO2 preferentially locates inside the PET droplets and hpho at both the interface and PP matrix. HphoM also locates within the PP matrix and at the interface, but large loadings (12%) can completely cover the surfaces of the droplets forming a physical barrier that avoids coalescence, leading to the formation of smaller droplets. A good correlation is found between the crystallization rate of PET (determined by DSC) and nanoparticles location, where hphi TiO2 induces the highest PET crystallization rate. PET lamellar morphology (revealed by TEM) is also dependent on particle location. The mechanical behavior improves in the elastic regime with TiO2 addition, but the plastic deformation of the material is limited and strongly depends on the type of TiO2 employed.

Targeted Release of Probiotics from Enteric Microparticulated Formulations.

The development of advanced probiotic delivery systems, which preserve bacteria from degradation of the gastrointestinal tract and achieve a targeted release mediated by pH-independent swelling, is of great interest to improve the efficient delivery of probiotic bacteria to the target tissue. Gram-positive and Gram-negative bacteria models (Lactobacillus acidophilus (Moro) Hansen and Mocquot (ATCC® 4356™) and Escherichia coli S17, respectively) have been successfully encapsulated for the first time in pH-independent microparticulate polymethacrylates (i.e., Eudraguard biotic) used for the targeted delivery of nutraceuticals to the colon. These bacteria have also been encapsulated within the mucoadhesive polymethacrylate Eudragit RS 100 widely used as targeted release formulation for active pharmaceutical ingredients. The enteric microparticles remained unaltered under simulated gastric conditions and released the contained viable microbial cargo under simulated intestinal conditions. Buoyancies of 90.2% and 57.3% for Eudragit and Eudraguard microparticles, respectively, and long-term stability (5 months) for the encapsulated microorganisms were found. Cytotoxicity of the microparticles formulated with both polymers was evaluated (0.5-20 mg/mL) on Caco-2 cells, showing high cytocompatibility. These results underline the suitability of the synthesized materials for the successful delivery of probiotic formulations to the target organ, highlighting for the first time the potential use of Eudraguard biotic as an effective enteric coating for the targeted delivery of probiotics.

Enzyme structure and function protection from gastrointestinal degradation using enteric coatings.

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.