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Refereed/Peer-reviewed Chronic Pseudomonas aeruginosa wound infections are highly prevalent and often untreatable due to biofilm formation, resulting in high antimicrobial tolerance. Standard antibiotic therapy for P. aeruginosa infections involves tobramycin, yet it is highly ineffective as monotherapy as tobramycin cannot penetrate the biofilm to elicit its antimicrobial effect. Lipid liquid crystal nanoparticles (LCNPs) have previously been shown to increase the antimicrobial efficacy and penetration of tobramycin against P. aeruginosa biofilms in vitro and ex vivo. Here, for the first time, we have developed a chronic P. aeruginosa biofilm infection in full-thickness wounds in mice to examine the potential of LCNPs to improve the effect of tobramycin, preclinically. After three doses, administered once a day, tobramycin-LCNPs significantly reduced the P. aeruginosa bacterial load in murine wounds 1000-fold more than unformulated tobramycin, which in turn showed no significant difference to the saline control treatment. Consistent with the improved P. aeruginosa eradication, the tobramycin-LCNPs promoted wound healing. In comparison to previous in vitro and ex vivo data, we show a strong in vitro-in vivo correlation between P. aeruginosa biofilm infection models. The enhanced activity of tobramycin-LCNPs in vivo in the preclinical murine model demonstrates the strong potential of LCNPs as a next-generation formulation approach to improve the efficacy of tobramycin against P. aeruginosa biofilm wound infections.

In the advent of the post-antibiotic era, new strategies are urgently required to improve the efficacy of antimicrobials and outsmart multi-drug resistant bacteria. Exploiting a basic survival mechanism of bacteria, lipase production, monoolein liquid crystal nanoparticles (MO-LCNPs) were investigated as a bacterial-triggered drug delivery system for three different antimicrobial compounds and compared with model sn-1/3 regiospecific and non-regiospecific lipases via pH-stat titration, proton nuclear magnetic resonance and in situ synchrotron small-angle X-ray scattering. The release of model hydrophobic (rifampicin) and macromolecular (alginate lyase) antimicrobials were triggered from MO-LCNPs at 82-fold and 7-fold higher rates (respectively) due to bacterial lipase digestion of MO-LCNPs, which could not be stimulated with a small hydrophilic antibiotic (ciprofloxacin HCl) or non-digestible, phytantriol-LCNPs. While sn-1/3 regiospecific lipase rapidly digested MO-LCNPs in a two-phase process, the single-phase digestion kinetics of the non-regiospecific lipase steadily digested the cubic Im3m structure and gave rise to lamellar structures that ultimately stimulated the triggered antibiotic release. Accordingly, MO-LCNPs have an application for localised Pseudomonas aeruginosa and Staphylococcus aureus infections that produce non-regiospecific lipases and for concentration-dependent antibiotics that have macromolecular (MW ~ 30 kDa) or hydrophobic (logP ~ 4) chemistries, as a triggered bolus release would be clinically efficacious for improved bacterial eradication. Refereed/Peer-reviewed

Refereed/Peer-reviewed Bacterial biofilm infections tolerate high concentrations of antibiotics and are insidiously challenging to treat. Liquid crystal nanoparticles (LCNPs) advance the efficacy of tobramycin in biofilm-related infections by increasing the penetration of antibiotics across the biofilm matrix. Herewith, we develop the LCNPs as a platform technology, demonstrating that the LCNPs can increase the efficacy of two antibiotic classes (i.e. aminoglycosides and colistin) in P. aeruginosa biofilm infections. In C. elegans, the LCNPs potentiated the antimicrobial effect and significantly improved the survival of the nematodes. In mice with a full-thickness excisional wound, LCNPs were non-toxic and did not impair wound repair. Compared to the unformulated antibiotic treatment, tobramycin-LCNPs reduced the chronic bacterial load by 100-fold in the wound. This was also emulated in an ex vivo P. aeruginosa porcine wound infection model. The LCNPs represent a versatile platform technology that improves the efficacy of cationic antibiotics against biofilm infections utilizing multiple administration routes.

It would be apt to say that one of the greatest accomplishments in modern medicine has been the development of vaccines against COVID-19, which had paralyzed the entire world for more than a year. Pfizer and BioNTech codeveloped the first COVID-19 vaccine that was granted emergency-use authorization or conditional approval in several regions globally. This article is an attempt to go 'behind-the-scenes' of this development process and highlight key factors that allowed us to move with this unprecedented speed, while adhering to normal vaccine-development requirements to generate the information the regulatory authorities needed to assess the safety and effectiveness of a vaccine to prevent an infectious disease, including quality and manufacturing standards. This is also a story of how Pfizer and BioNTech leveraged our combined skill sets and experience to respond to the global health crisis to progress this program swiftly while ensuring the compliance with our high-quality standards and keeping patient safety at the forefront. We will also highlight multiple other factors that were instrumental in our success.

Staphylococcal biofilms cause many infectious diseases and are highly tolerant to the effects of antimicrobials;this is partly due to the biofilm matrix, which acts as a physical barrier retarding the penetration and reducing susceptibility to antimicrobials, thereby decreasing successful treatment outcomes. In this study, both single and mixed micellar systems based on poly vinyl caprolactam (PCL)-polyethylene glycol (PEG) copolymers were optimised for delivery of chlorhexidine (CHX) to S. aureus, MRSA and S. epidermidis biofilms and evaluated fortheir toxicity using Caenorhabditis elegans. The respective polyethylene glycol (PEG) and poly vinyl caprolactam(PCL) structural components promoted stealth properties and enzymatic responsive release of CHX inside biofilms,leading to significantly enhanced penetration (56%) compared with free CHX and improving the efficacy against Staphylococcus aureus biofilms grown on an artificial dermis (2.4 log reduction of CFU). Mixing Soluplus-based micelles with Solutol further enhanced the CHX penetration (71%) and promoted maximumr eduction in biofilm biomass (> 60%). Nematodes-based toxicity assay showed micelles with no lethal effects as indicated by their high survival rate (100%) after 72 h exposure. This study thus demonstrated that bio-responsivecarriers can be designed to deliver a poorly water-soluble antimicrobial agent and advance the controlof biofilm associated infections. Refereed/Peer-reviewed

The glycoside hydrolase, PslG, attacks and degrades the dominant Psl polysaccharide in the exopolymeric substance (EPS) matrix of Pseudomonas aeruginosa biofilms and is a promising therapy to potentiate the effect of antibiotics. However, the need for coadministration with an antibiotic and the potential susceptibility of PslG to proteolysis highlights the need for an effective delivery system. Here, we compared liposomes versus lipid liquid crystal nanoparticles (LCNPs) loaded with PslG and tobramycin as potential formulation approaches to (1) protect PslG from proteolysis, (2) trigger the enzyme’s release in the presence of bacteria, and (3) improve the total antimicrobial effect in vitro and in vivo in a Caenorhabditis elegans infection model. LCNPs were an effective formulation strategy for PslG and tobramycin that better protected the enzyme against proteolysis, triggered and sustained the release of PslG, improved the antimicrobial effect by 10–100-fold, and increased the survival of C. elegans infected with P. aeruginosa. Digestible LCNPs had the advantage of triggering the enzyme’s release in the presence of bacteria. However, compared to nondigestible LCNPs, negligible differences arose between the LCNPs' ability to protect PslG from proteolysis and potentiate the antimicrobial activity in combination with tobramycin. In C. elegans, the improved antimicrobial efficacy was comparable to tobramycin-LCNPs, although the PslG + tobramycin-LCNPs achieved a greater than 10-fold reduction in bacteria compared to the unformulated combination. Herewith, LCNPs are showcased as a promising protective delivery system for novel biofilm dispersing enzymes combined with antibiotics, enabling infection-directed therapy and improved performance. Refereed/Peer-reviewed

Pseudomonas aeruginosa biofilms cause persistent and chronic infections, most known clinically in cystic fibrosis (CF). Tobramycin (TOB) is a standard anti-pseudomonal antibiotic; however, in biofilm infections, its efficacy severely decreases due to limited permeability across the biofilm matrix. Herewith, a biomimetic, nanostructured, lipid liquid crystal nanoparticle-(LCNP)-formulation is discovered to significantly enhance the efficacy of TOB and eradicate P. aeruginosa biofilm infections. Using an advanced, biologically-relevant co-culture model of human CF bronchial epithelial cells infected with P. aeruginosa biofilms at an air–liquid interface, nebulized TOB-LCNPs completely eradicated 1 × 109 CFU mL−1 of P. aeruginosa after two doses, a 100-fold improvement over the unformulated antibiotic. The enhanced activity of TOB is not observed with a liposomal formulation of TOB or with ciprofloxacin, an antibiotic that readily penetrates biofilms. It is demonstrated that the unique nanostructure of the LCNPs drives the enhanced penetration of TOB across the biofilm barrier, but not through the healthy lung epithelium barrier, significantly increasing the available antibiotic concentration at the site of infection. The LCNPs are an innovative strategy to improve the performance of TOB as a directed pulmonary therapy, enabling the administration of lower doses, reducing the toxicity, and amplifying the anti-biofilm activity of the anti-pseudomonal antibiotic. Refereed/Peer-reviewed

The major pathogen found in the lungs of adult cystic fibrosis (CF) patients is Pseudomonas aeruginosa, which builds antibiotic-resistant biofilms. Pulmonary delivery of antibiotics by inhalation has already been proved advantageous in the clinic, but the development of novel anti-infective aerosol medicines is complex and could benefit from adequate in vitro test systems. This work describes the first in vitro model of human bronchial epithelial cells cultivated at the air–liquid interface (ALI) and infected with P. aeruginosa biofilm and its application to demonstrate the safety and efficacy of aerosolized anti-infective nanocarriers. Such a model may facilitate the translation of novel therapeutic modalities into the clinic, reducing animal experiments and the associated problems of species differences. A preformed biofilm of P. aeruginosa PAO1 was transferred to filter-grown monolayers of the human CF cell line (CFBE41o-) at ALI and additionally supplemented with human tracheobronchial mucus. This experimental protocol provides an appropriate time window to deposit aerosolized ciprofloxacin-loaded nanocarriers at the ALI. When applied 1 h post-infection, the nanocarriers eradicated all planktonic bacteria and reduced the biofilm fraction of the pathogen by log 6, while CFBE41o- viability and barrier properties were maintained. The here described complex in vitro model approach may open new avenues for preclinical safety and efficacy testing of aerosol medicines against P. aeruginosa lung infection. Graphical abstract

Biofilm-dispersing enzymes degrade the extracellular polymeric matrix surrounding bacterial biofilms, disperse the microbial community and increase their susceptibility to antibiotics and immune cells. Challenges for the clinical translation of biofilm-dispersing enzymes involve their susceptibility to denaturation, degradation, and clearance upon administration in vivo. Drug delivery systems aim to overcome these limitations through encapsulation, stabilization and protection from the exterior environment, thereby maintaining the enzymatic activity. Smart drug delivery systems offer target specificity, releasing payloads at the site of infection while minimizing unnecessary systemic exposure. This review highlights critical advances of biofilm-dispersing enzymes as a novel therapeutic approach for biofilm-associated infections. We explore how smart, bio-responsive delivery systems overcome the limiting factors of biofilm-dispersing enzymes and summarize the key systems designed. This review will guide future developments, focusing on utilizing selective and specific therapies in a targeted fashion to meet the unmet therapeutic needs of biofilm infections. Refereed/Peer-reviewed

Bacterial infections are an imminent global healthcare threat evolving from rapidly advancing bacterial defence mechanisms that antibiotics fail to overcome. Antibiotics have been designed for systemic administration to target planktonic bacteria, leading to difficulties in reaching the site of localized bacterial infection and an inability to overcome the biological, chemical and physical barriers of bacteria, including biofilms, intracellular infections and antimicrobial resistance. The amphiphilic, biomimetic and antimicrobial properties of lipids provide a promising toolbox to innovate and advance antimicrobial therapies, overcoming the barriers presented by bacteria in order to directly and effectively treat recalcitrant infections. Nanoparticulate lipid-based drug delivery systems can enhance antibiotic permeation through the chemical and physical barriers of bacterial infections, as well as fuse with bacterial cell membranes, release antibiotics in response to bacteria and act synergistically with loaded antibiotics to enhance the total antimicrobial efficacy. This review explores the barriers presented by bacterial infections that pose bio-pharmaceutical challenges to antibiotics and how different structural and functional mechanisms of lipids can enhance antimicrobial therapies. Different nanoparticulate lipid-based systems are presented as valuable drug delivery systems to advance the efficacy of antibiotics, including liposomes, liquid crystalline nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers and lipid nanocarriers. In summary, liquid crystalline nanoparticles are emerging with the greatest potential for clinical applications and commercial success as an “all-rounder” advanced lipid-based antimicrobial therapy that overcomes the multiple biological, chemical and physical barriers of bacteria. Refereed/Peer-reviewed

Bacterial biofilms account for up to 80% of all communityacquired infections for which bacterial eradication is currently not achievable using conventional antimicrobial treatments. The protective matrix that engulfs biofilm-associated bacteria frequently renders antibiotics ineffective. Glycoside hydrolases are a class of enzymes that break down the biofilm matrix, thereby increasing the effectiveness of antibiotics. Herein, nanostructured liquid crystals composed of glyceryl monooleate (GMO) were investigated as an infection responsive delivery system for alginate lyase (glycoside hydrolase) and gentamicin (antibiotic) to treat Pseudomonas biofilms. The presence of Pseudomonas lipase triggered the release of alginate lyase and gentamicin from the GMO liquid crystals. Treatment with the liquid crystals containing alginate lyase and gentamicin resulted in a greater than 2-log reduction in mucoid Pseudomonas aeruginosa (clinical isolate) biofilm. The anti-biofilm activity of alginate lyase and gentamicin from the liquid crystals was sustained for 2 days and equivalent to the respective unformulated solution treatments. Accordingly, GMO based liquid crystals are a promising responsive delivery system for alginate lyase and gentamicin to combat topical Pseudomonas infections Refereed/Peer-reviewed

Bacterial biofilms are associated with a number of recurring infectious diseases and are a majorcause for antibiotic resistance. Despite the broad use of polymeric microparticles and nanoparticles in biomedical research it is not clear which particle size is more effective against biofilms. The purpose of this study was to evaluate the efficacy of sustained release poly-lactic-co-glycolic acid(PLGA) micro- and nanoparticles containing ciprofloxacin against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa The PLGA particles were prepared by the double emulsion solvent evaporation method. The resulting microparticles (12 μm) and nanoparticles (300 nm) contained drug loads of 7.3% and 4.5%(w/w) ciprofloxacin, respectively. Drug release was complete within one week following comparable release profiles for both particle sizes. Micro- and nanoparticles demonstrated a similar in vitro antibiofilm performance against mature P. aeruginosa and S. aureus with marked differences between the two strains. The sustained release of ciprofloxacin from micro- and nanoparticles over 6 days was equally effective as the continuous treatment with ciprofloxacin solution over the same period resulting in the eradication of culturable S. aureus suggesting that reformulation of ciprofloxacin as sustained release PLGA micro- and nanoparticles might be valuable formulation approaches for the treatment of biofilms. Refereed/Peer-reviewed