Nanocarrier-formulated antimicrobials and microfluidics-based screening assays

Antibiotics and other antimicrobial agents have allowed the successful treatment of a range of infectious diseases. However, due to their extensive use a range of bacteria have developed multiple resistances to many antibiotics at therapeutically acceptable doses. In this thesis, one possible solution to overcome antimicrobial resistance was presented. This approach is based on the development of nanocarrier-formulated antimicrobial agents which have been encapsulated into biodegradable nanoparticles (NPs). A novel type of very efficient nanocarrier based on shellac; natural polymeric material of insect origin was designed. This nanocarrier was used to encapsulate and deliver antibiotics or antimicrobial agents such as berberine chloride (BRB), chlorhexidine di-gluconate (CHX), curcumin (CUR), and vancomycin hydrochloride (VCM). The nanocarrier was formulated and loaded with antimicrobial agent in two steps: (i) The first step involved controlled precipitation of aqueous ammonium shellac salts by a simultaneous pH change and adsorption of surface active polymer (Poloxamer 407) in the presence of the active antimicrobial component. In this step, simultaneously drug-loaded shellac nanoparticles was formed and coated them with a sterically stabilizing polymer, which can be allowed to maintain their stability and ensure long shelf-life. Stable shellac nanoparticles were produced at pH 5 with a particle hydrodynamic diameter of 66±5 nm with zeta potential – 18±8 mV. (ii) The second step involved charge-reversing the produced shellac nanoparticles by doping with insoluble cationic surfactant (ODTAB), which gave them a positive surface charge in order to promote the nanocarrier adhesion to the negatively charged cell membranes of typical bacterial cells. Physical and chemical parameters such as the effect of different concentrations of the surface active polymer as well as the berberine, chlorhexidine, curcumin and vancomycin concentrations were studied on the size distribution of the produced nanoparticles and their zeta potential.Optimal nanocarrier stability was obtained at a fixed ratio of (0.25:0.2) wt.% of shellac : Poloxamer 407 concentrations. Using 0.01 wt.%-0.07 wt.% concentration range of BRB, CHX, CUR and VCM with 0.25 wt.% shellac at pH 5 to be encapsulated within shellac NPs. The maximum encapsulation efficiencies of 60%, 92%, 100% and 87.5% for BRB, CHX, CUR, and VCM, respectively, were achieved. The release profiles of BRB, CHX, CUR and VCM loaded the developed shellac nanocarriers at pH 5.5 and pH 7.4 were studied, and sustained release from the formulations was confirmed upon dilution over a period. TEM images revealed that the NPs and the formulated antimicrobial nanoparticles have a spherical shape and agree with the DLS (zetasizer) measurements. The interaction between the NPs and the antimicrobials was characterized using FTIR and UV-visible techniques.The importance of the nanocarrier architecture on the antimicrobial activity of the loaded agent was studied. The antimicrobial activity of BRB-, CHX-, CUR-, and VCM- loaded shellac nanocarriers was studied upon incubation with microalgae, yeast, and E.coli at different incubation times. Although the free BRB, CHX, CUR and VCM in aqueous solution showed significant antimicrobial effect on these microorganisms, they showed weaker antimicrobial action when they were encapsulated within shellac NPs without doping with ODTAB. This reduction in activity was due to the repulsion between the negatively charged shellac NPs and the negative cell membrane which did not allow the encapsulated antimicrobials to be released near the cell wall vicinity. In addition to this, the attraction between the cationic antimicrobial agent and the shellac NPs led to slower the drug release. However, upon functionalization of the loaded-shellac NPs with a cationic surfactant ODTAB, their surface charge changed from negative to positive. Optimum conditions were found where the nanocarriers become cationic and still maintained their stability due to steric interactions. Consequently, the antimicrobial activity of these ODTAB-coated shellac NPs loaded with antimicrobial agents showed a significantly higher antimicrobial effect than the equivalent overall concentration of the free antimicrobials in solution. This effect was due to the strong electrostatic adhesion with the cell membrane which allowed the antimicrobial agents to be released directly into the microbial cell. Hence the cationically functionalized nanocarrier provides a boost of the antimicrobial action of the loaded agent.A microfluidic device for cell trapping was also designed which is suitable for microscreening cell based assay. The microfluidic composed of two layers; a top layer of PDMS which contains connecting tubing, and the bottom layer was a microscope glass with two inlet channels, microchamber, and one outlet channels. Microbial cells trapped inside the microchamber with the antimicrobial agents was tested by using magnetic beads as a chamber “gate keeper” to trap the cells inside the microchamber and allow the fluids and the tested formulation to pass through the outlet channel. This is expected to lead further development of high throughput systems for testing antimicrobial agents on a range of microbial cells