Advanced nanotechnologies for overcoming antimicrobial resistance

Multidrug-resistant pathogens are prevalent in chronic wounds. There is an urgent need to develop novel antimicrobials and formulation strategies to overcome antibiotic resistance and provide a safe alternative to traditional antibiotics. Chapter 2 aims to create a novel nanocarrier for two cationic antibiotics, tetracycline hydrochloride and lincomycin hydrochloride, overcoming antibiotic resistance. In this study, the use of surface-functionalised polyacrylic copolymer nanogels as carriers for cationic antibiotics is investigated. 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. The data reveals that bPEI-coated nanogels with encapsulated tetracycline or lincomycin displayed increased antimicrobial performance against selected wound-derived bacteria, including strains resistant to the free antibiotic in solution.Next, after experimentation into the use of Carbopol nanogels against antibiotic-resistant wound-derived pathogens, in planktonic form, the work in chapter 3 investigated their use against biofilm-formed pathogens. 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. Novel nanotechnology is applied to do this, based on protease-functionalised nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with the protease Alcalase, "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 defences. This thesis's work demonstrates that they are effective against six wound biofilm-forming bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis. Additionally, it is shown 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 hours of treatment.Chapter 4 combines the work of chapter 2 (NPs against antibiotic-resistant pathogens) and chapter 3 (NPs against biofilm-forming pathogens). This concept is demonstrated by encapsulating Penicillin G and Oxacillin into shellac nanoparticles, subsequently coated Alcalase. It is shown for the first time that these active nanocarriers can destroy biofilms of S. aureus resistant to Penicillin G and are significantly more effective in killing the bacterial cells within compared to an equivalent concentration of free antibiotic. The approach of concentrating the antibiotic by encapsulating it into a nanocarrier allows a localised antibiotic delivery to the anionic cell wall, facilitated by coating the NPs with a cationic protease. This approach allowed the antibiotic to restore its effectiveness against S. aureus, characterised as resistant to the same antibiotic and cause rapid bacterial biofilm degradation. This approach could be potentially used to revive old antibiotics which have already limited clinical use due to developed resistance.Chapter 5 continued investigating the antimicrobial properties of antibiotic-loaded shellac NPs, with a cationic protease surface functionalisation, however this time on a pathogen fungal species, Candida albicans. These Amphotericin B (AmpB)‐loaded shellac NPs are fabricated by pH‐ induced nucleation of aqueous solutions of shellac and AmpB in the presence of Poloxamer 407 (P407) as a steric stabiliser (in the same fashion as penicillin G and oxacillin in chapter 4. The AmpB‐loaded shellac NPs are surface coated with the cationic protease Alcalase. The AmpB‐loaded shellac NPs show a remarkable boost of their antifungal action compared to free AmpB when applied to C. albicans in both planktonic and biofilm forms. The surface functionalisation with a cationic protease allows the NPs to adhere to the fungal cell walls, delivering AmpB directly to their membranes. Additionally, the hydrolysing activity of the protease coating degrades the biofilm matrix, thus increasing the effectiveness of the encapsulated AmpB compared to free AmpB at the same concentration.Additionally, these protease‐coated nanocarrier-based antibiotics showed no detectable cytotoxic effect against human keratinocytes. It is envisaged these antibiotic-loaded NPs. Subsequently, surface functionalised with the cationic protease could be potentially used to treat antibiotic-resistant biofilm infections in the clinic, for example, in recalcitrant chronic wounds. Chapter 6 outlines future work which could be performed using these NP formulations