ULTRASOUND-TRIGGERED NANO-FORMULATIONS FOR PREVENTION AND TARGETING OF BACTERIAL BIOFILMS

Abstract

Prior to the antibiotic era, bacterial infections were the leading cause of death. Antibiotics were developed to cure a variety of ailments and saved many lives. However, due to overuse and inappropriate use of antibiotics, the emergence of bacterial resistance has expedited the so-called "post-antibiotic age." As a result, increasing death rates due to resistant bacterial infections a significant threat to public health and the world economies. The ability of many bacterial strains to form biofilms is the leading cause of the high viability and spread of the bacteria in tissues and chronic wounds. Modern antibiotics have shown low effectiveness due to the impenetrable bacteria extra polymer substance (EPS) of the biofilm. In the present thesis we explore a new strategy to target and prevent formation of drug-resistant bacterial biofilm infections. We developed study, novel shellac/enzyme nanoparticle formulations of three different enzymes (Alcalase, Savinase, Cellulase) and explored their ability to target biofilms of S. epidermidis. Our idea is based on preventing the premature release of the enzyme which can degrade the biofilm by depositing a silica shell over the nanocarrier particles that covers the surface of sterically stabilized shellac core with encapsulated enzyme which plays a role in the stimulus-triggered release of an active component to eradicate bacterial biofilms. We assessed the quality of the formed silica shells using dynamic light scattering, Transmission Electron Microscopy (TEM) and Energy-dispersive X-ray spectroscopy. TEM imaging allowed to assess the thickness of the silica shell. We tested both uncoated and silica coated shellac/enzyme nanoparticles in their effectiveness to degrade the bacterial biofilms. The most efficient nano-formulations were TEOS-coated Cellulase-loaded shellac nanoparticles which showed significant decrease in the residual biofilm biomass of S. epidermidis bacteria strain. We observed a significant increase of the enzyme release from silica-coated enzyme nanocarriers upon application of ultrasonic trigger, compared with non-coated enzyme nanocarrier. Most of the additional amount of enzyme was released during and very shortly after sonication. This demonstrated a successful ultrasonic trigger for the particles in the biofilm. We also evaluated the cytotoxicity of these formulations where the results indicated only a modest cytotoxicity on HeLa cell line which were used as a proxy for human cells. We also did the same encapsulation by using (3-Aminopropyl)triethoxysilane (APTES) which lead to similar results, but apparently higher cytotoxicity towards Hela cells. The developed silicaencapsulated enzyme nanocarriers have the potential to prevent and target bacterial biofilm by pretreating surfaces prone to bacterial biofilm infection and triggering the enzyme release by application of medical grade ultrasound. The treatment of chronic wounds with such silica shell-encapsulated enzyme-loaded nanoparticles could help develop more efficient ways of removal of bacterial biofilm by using non-invasive techniques to improve patient outcomes.