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.

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.

Colistin-loaded aerosolizable particles for the treatment of bacterial respiratory infections.

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.

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.

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.

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.

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.