Ultrasound-enhanced drug delivery has shown great promise in providing targeted burst release of drug at the site of the disease. Yet current solid ultrasound-responsive particles are non-degradable with limited potential for drug-loading. Here, we report on an ultrasound-responsive multi-cavity poly(lactic-co-glycolic acid) microparticle (mcPLGA MP) loaded with rhodamine B (RhB) with or without 4′,6-diamidino-2-phenylindole (DAPI) to represent small molecule therapeutics. After exposure to high intensity focused ultrasound (HIFU), these delivery vehicles were remotely implanted into gel and porcine tissue models, where the particles rapidly released their payload within the first day and sustained release for at least seven days. RhB-mcPLGA MPs were implanted with HIFU into and beyond the sub-endothelial space of porcine arteries without observable damage to the artery. HIFU also guided the location of implantation; RhB-mcPLGA MPs were only observed at the focus of the HIFU away from the direction of ultrasound. Once implanted, DAPI co-loaded RhB-mcPLGA MPs released DAPI into the arterial wall, staining the nucleus of the cells. Our work shows the potential for HIFU-guided implantation of drug-loaded particles as a strategy to improve the local and sustained delivery of a therapeutic for up to two weeks.
12Biofilm monitoring in environmental and biomedical applications remains a challenge. 13 Conventional biochemical methods do not yet provide a quick quantitative measure of 14 attached biomass. Thus, there is a need for rapid in situ detection tools for routine biofilm 15 characterization. Electrochemical impedance spectroscopy (EIS) characterizes the 16 electroactivity of bacteria within a biofilm and has been extensively used to monitor strong 17 electroactive biofilms. Yet, studies on weak electricigens such as Pseudomonas aeruginosa 18 remain underrepresented. Here, conductive indium tin oxide coated polyethylene 19 terephthalate (ITO:PET) sheets were used as flexible growth substrates instead of more 20 conventional carbonaceous or gold materials. EIS was compared with standard optical 21 methods for the detection of P. aeruginosa biofilms formed on ITO:PET under static growth 22 conditions. Relaxation time analysis showed a dominant time constant at approximately 1 23
Bacterial biofilms are typically more tolerant to antimicrobials compared to bacteria in the planktonic phase and therefore require alternative treatment approaches. Mechanical biofilm disruption from ultrasound may be such an alternative by circumventing rapid biofilm adaptation to antimicrobial agents. Although ultrasound facilitates biofilm dispersal and may enhance the effectiveness of antimicrobial agents, the resulting biological response of bacteria within the biofilms remains poorly understood. To address this question, we investigated the microstructural effects of Pseudomonas aeruginosa biofilms exposed to high intensity focused ultrasound (HIFU) at different acoustic pressures and the subsequent biological response. Confocal microscopy images indicated a clear microstructural response at peak negative pressures equal to or greater than 3.5 MPa. In this pressure amplitude range, HIFU partially reduced the biomass of cells and eroded exopolysaccharides from the biofilm. These pressures also elicited a biological response; we observed an increase in a biomarker for biofilm development (cyclic-di-GMP) proportional to ultrasound induced biofilm removal. Cyclic-di-GMP overproducing mutant strains were also more resilient to disruption from HIFU at these pressures. The biological response was further evidenced by an increase in the relative abundance of cyclic-di-GMP overproducing variants present in the biofilm after exposure to HIFU. Our results, therefore, suggest that both physical and biological effects of ultrasound on bacterial biofilms must be considered in future studies.
This thesis is a conclusion of a tremendous learning experience both professionally and personally throughout the four years at NTU. This would not be possible without the exceptional mentorship and support from my supervisor, collaborators and peers. I am indebted to my supervisor Assoc. Prof. James Kwan for providing me the amazing opportunity to join his research group and work on such an interesting project. This work would not have been possible without his exemplary guidance, constant support and encouragement. He introduced me to a new perspective of research and for that, I am truly grateful. I also take this opportunity to express my profound gratitude to Assoc. Prof. Enrico Marsili for sharing his invaluable time and extensive knowledge in biofilm electrochemistry. I am thankful to Assoc. Prof. Scott Rice for his crucial insights in the microbiological part of the work and Assoc. Prof. Manojit Pramanik for guiding me through the final months before PhD completion.I am grateful to Dr. Umesh Jonnalagadda for investing his time and effort to guide me through signal processing and coding component of the project. Moreover, thanks to the tireless and dedicated FYP students Mr.
Bacterial infections are increasingly difficult to treat due to their growing resistance to antibiotics. Most of these bacterial infections form a biofilm that limits the effectiveness of the antibiotic. Biofilms are microbial cells that are protected by a self-generated matrix of extracellular polymeric substances. In addition to their intrinsic antibiotic resistance, these biofilms are able to respond to the stresses from the antibiotic by inducing drug resistance mechanisms. Currently, the strategy to combat drug resistance is to develop novel drugs, however, the rate of drug development is being surpassed by the rate of drug resistance. There is therefore a need for alternative means in enhancing the efficacy of current drug therapeutics. We propose to use of high intensity focused ultrasound (HIFU) to disrupt the biofilm and promote drug penetration. However, the effects of HIFU on these bacterial communities remain unknown. Here we report on microstructural changes within biofilms formed by Pseudomonas aeruginosa due to exposure to HIFU at 500 kHz center frequency. Changes to the biofilm were nondestructively measured through impedance spectroscopy and confocal microscopy. Biofilms were shown to induce cavitation (as measured by a passive cavitation detector) at relatively low pressure amplitudes suggesting the presence of cavitation nuclei within the extracellular matrix.
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