Overcoming Beta-Lactamase-Based Antimicrobial Resistance by Nanocarrier-Loaded Clavulanic Acid and Antibiotic Cotreatments.

Antimicrobial resistance (AMR) is one of the major threats to modern healthcare. Many types of bacteria have developed resistance to multiple antibiotic treatments, while additional antibiotics have not been recently brought to market. One approach to counter AMR based on the beta-lactamase enzyme has been to use cotreatments of an antibiotic and an inhibitor, to enhance the antibiotic action. Here, we aimed to enhance this technique by developing nanocarriers of two cationic beta-lactam class antibiotics, amoxicillin, and ticarcillin, combined with a beta-lactamase inhibitor, clavulanic acid, which can potentially overcome this type of AMR. We demonstrate for the first time that beta-lactamase inhibitor-loaded nanocarriers in cotreatments with either free or nanocarrier-loaded beta-lactam antibiotics can enhance their effectiveness further than when used alone. We use surface-functionalized shellac-/Poloxamer 407-stabilized antibiotic nanocarriers on Pseudomonas aeruginosa, which is susceptible to ticarcillin but is resistant to amoxicillin. We show an amplification of the antibiotic effect of amoxicillin and ticarcillin loaded in shellac nanoparticles, both alone and as a cotreatment with free or nanocarrier-loaded clavulanic acid. We also report a significant increase in the antimicrobial effects of clavulanic acid loaded in such nanocarriers as a cotreatment. We explain the increased antimicrobial activity of the cationically functionalized antibiotic-loaded nanoparticles with electrostatic attraction to the bacterial cell wall, which delivers higher local antibiotic and inhibitor concentrations. The effect is due to the accumulation of the clavulanic acid-loaded nanocarriers on the bacterial cell walls that allows a higher proportion of the inhibitor to engage with the produced intracellular beta-lactamases. These nanocarriers were also found to have a very low cytotoxic effect against human keratinocytes, which shows great potential for overcoming enzyme-based AMR.

Enhanced clearing of Candida biofilms on a 3D urothelial cell in vitro model using lysozyme-functionalized fluconazole-loaded shellac nanoparticles.

Candida urinary tract biofilms are increasingly witnessed in nosocomial infections due to reduced immunity of patients and the hospital ecosystem. The indwelling devices utilized to support patients with urethral diseases that connect the unsterilized external environment with the internal environment of the patient are another significant source of urinary tract biofilm infections. Recently, nanoparticle (NP)-associated therapeutics have gained traction in a number of areas, including fighting antibiotic-resistant bacterial biofilm infection. However, most studies on nanotherapeutic delivery have only been carried out in laboratory settings rather than in clinical trials due to the lack of precise in vitro and in vivo models for testing their efficiency. Here we develop a novel biofilm-infected 3D human urothelial cell culture model to test the efficiency of nanoparticle (NP)-based antifungal therapeutics. The NPs were designed based on shellac cores, loaded with fluconazole and coated with the cationic enzyme lysozyme. Our formulation of 0.2 wt% lysozyme-coated 0.02 wt% fluconazole-loaded 0.2 wt% shellac NPs, sterically stabilised by 0.25 wt% poloxamer 407, showed an enhanced efficiency in removing Candida albicans biofilms formed on 3D layer of urothelial cell clusteroids. The NP formulation exhibited low toxicity to urothelial cells. This study provides a reliable in vitro model for Candida urinary tract biofilm infections, which could potentially replace animal models in the testing of such antifungal nanotechnologies. The reproducibility and availability of a well-defined biofilm-infected 3D urothelial cell culture model give valuable insights into the formation and clearing of fungal biofilms and could accelerate the clinical use of antifungal nanotherapeutics.

Biofilm-Infected Human Clusteroid Three-Dimensional Coculture Platform to Replace Animal Models in Testing Antimicrobial Nanotechnologies.

Microbial biofilms are a major concern in wound care, implant devices, and organ infections. Biofilms allow higher tolerance to antimicrobial drugs, can impair wound healing, and potentially lead to sepsis. There has been a recent focus on developing novel nanocarrier-based delivery vehicles to enhance the biofilm penetration of traditional antibacterial drugs. However, a feasible in vitro human skin model to mimic the biofilm formation and its treatment for clearance have not yet been reported. This study describes the benefits of using an innovative bacterial biofilm-infected keratinocyte clusteroid model for the first time. It paves a new way for testing innovative nanomedicine delivery systems in a rapid and reproducible way on a realistic human cell-based platform, free of any animal testing. Herein, we have developed a novel composite 3D biofilm/human keratinocyte clusteroid coculture platform, which was used to measure biofilm clearance efficiency of nanoparticle (NP)-based therapeutics. We tested this model by treating the biofilm-infected 3D coculture layers by a ciprofloxacin-loaded Carbopol nanogel particles, surface-functionalized by the cationic protease Alcalase. We measured the antibacterial efficiency of the NP treatment on clearing Staphylococcus aureus and Pseudomonas aeruginosa biofilms on the 3D keratinocyte clusteroid/biofilm coculture model. Our experiments showed that these bacteria can infect the 3D layer of keratinocyte clusteroids and produce a stable biofilm. The biofilms were efficiently cleared by treatment with a formulation of 0.0032 wt % ciprofloxacin-loaded in 0.2 wt % Carbopol NPs surface-functionalized with 0.2 wt % Alcalase. Taken together, these promising results demonstrate that our coculture model can be exploited as a novel platform for testing the biofilm-eliminating efficiency of various NP formulations emulating skin and wound infections and could have wider applicability to replace animal models in similar experiments. This 3D cell culture-based platform could help in developing and testing of more effective antibacterial agents for clinical applications of antiplaque dental treatments, implants, infection control, and wound dressings.

Enhanced Clearing of Wound-Related Pathogenic Bacterial Biofilms Using Protease-Functionalized Antibiotic Nanocarriers.

Biofilms are prevalent in chronic wounds and once formed are very hard to remove, which is associated with poor outcomes and high mortality rates. Biofilms are comprised of surface-attached bacteria embedded in an extracellular polymeric substance (EPS) matrix, which confers increased antibiotic resistance and host immune evasion. Therefore, disruption of this matrix is essential to tackle the biofilm-embedded bacteria. Here, we propose a novel nanotechnology to do this, based on protease-functionalized nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with the protease Alcalase 2.4 L FG, "digest" their way through the biofilm EPS matrix, reach the buried bacteria, and deliver a high dose of antibiotic directly on their cell walls, which overwhelms their defenses. We demonstrated their effectiveness against six wound biofilm-forming bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis. We confirmed a 6-fold decrease in the biofilm mass and a substantial reduction in bacterial cell density using fluorescence, atomic force, and scanning electron microscopy. Additionally, we showed that co-treatments of ciprofloxacin and Alcalase-coated Carbopol nanogels led to a 3-log reduction in viable biofilm-forming cells when compared to ciprofloxacin treatments alone. Encapsulating an equivalent concentration of ciprofloxacin into the Alcalase-coated nanogel particles boosted their antibacterial effect much further, reducing the bacterial cell viability to below detectable amounts after 6 h of treatment. The Alcalase-coated nanogel particles were noncytotoxic to human adult keratinocyte cells (HaCaT), inducing a very low apoptotic response in these cells. Overall, we demonstrated that the Alcalase-coated nanogels loaded with a cationic antibiotic elicit very strong biofilm-clearing effects against wound-associated biofilm-forming pathogenic bacteria. This nanotechnology approach has the potential to become a very powerful treatment of chronically infected wounds with biofilm-forming bacteria.

Breathing new life into old antibiotics: overcoming antibacterial resistance by antibiotic-loaded nanogel carriers with cationic surface functionality.

Multidrug-resistant pathogens are prevalent in chronic wounds. There is an urgent need to develop novel antimicrobials and formulation strategies that can overcome antibiotic resistance and provide a safe alternative to traditional antibiotics. This work aimed to develop a novel nanocarrier for two cationic antibiotics, tetracycline hydrochloride and lincomycin hydrochloride which can potentially overcome antibiotic resistance. In this study, we report the use of surface functionalised polyacrylic copolymer nanogels as carriers for cationic antibiotics. These nanogels can encapsulate small cationic antimicrobial molecules and act as a drug delivery system. They were further functionalised with a biocompatible cationic polyelectrolyte, bPEI, to increase their affinity towards the negatively charged bacterial cell walls. These bPEI-coated nanocarrier-encapsulated antibiotics were assessed against a range of wound isolated pathogens, which had been shown through antimicrobial susceptibility testing (AST) to be resistant to tetracycline and lincomycin. Our data reveal that bPEI-coated nanogels with encapsulated tetracycline or lincomycin displayed increased antimicrobial performance against selected wound-derived bacteria, including strains highly resistant to the free antibiotic in solution. Additionally, our nanocarrier-based antibiotics showed no detectable cytotoxic effect against human keratinocytes. We attribute the increase in the antimicrobial activity of the cationically functionalised antibiotic-loaded nanogel carriers to specific electrostatic adhesion to the microbial cell wall delivering a higher local antibiotic concentration, confirmed by scanning electron microscopy. Such a nanotechnology based approach may enhance the effectiveness of a wide variety of existing antibiotics, offering a potentially new mechanism to overcome antibiotic resistance.