Studies on Antimicrobial Properties of Metals and Metal Oxide Based Nanostructures

Abstract

Infection is a biological process in which the host organism’s body tissues are invaded by disease causing microorganisms like virus, bacteria, fungi and other macroparasites. Infectious disease is the outcome of interplay between pathogenic microorganisms and immune system of the host. In the history of human civilization, the epidemics suffered by mankind are due to the infectious diseases. Billions of humans were killed due to infections which were not curable. According to the World Health Organization report in the year 2002, 26% of the total deaths were caused by the infectious diseases. Nearly 14.7 million people died due to infections in the year 2002. The mortality caused by infectious diseases is expected to rise further due to environmental pollution and rapid changes in the ecosystem. Anti-infective drugs are used in the curative and preventive health care to treat various infections caused by pathogenic microorganisms. An antibiotic is a substance that kills microorganisms or inhibits their growth. Antibiotics work on different mechanisms. Some of them kill the microorganisms, while the others just prevent their growth. Few antibiotics disrupt the bacterial cell walls or interfere with the genetic material of the cell, leading to death, while the others may prevent them from making important chemical compositions required for their growth. To fight against infections, antibiotic is the only option available with pharmacologists. Even though antibiotics are widely used, cautions must be exercised while using them. Some common side effects that occur in patients include nausea, diarrhea, stomach pains, headache, fever, body and muscle aches and vomiting to life threatening diseases like abnormal blood clotting, kidney stones, blood disorders, etc. Antibiotics can decrease the effectiveness of birth control pills. Hence, there is an urgent need for the development of alternative medicines, which are as efficient as antibiotics but have no potential side effects. Development of pathogenic strains of microorganisms that are resistant to most of the currently available antibiotics is another challenge in health care. For example, MRSA (methicillin resistant S. aureus) bacteria kill 5000 hospital patients a year in the UK alone. These strains are resistant to at least one antibiotic, which is presently available in the market and any method of attacking them not involving antibiotics, is becoming increasingly important. Microbes possess an internal mechanism of changing their structure so that the antibiotic no longer works. They develop ways to inactivate or neutralize the antibiotics. They can transfer gene coding for antibiotic resistance amongst them, making it possible for the microorganisms never exposed to an antibiotic to acquire resistance. Further, the development process of antibiotics is slow and lengthy and hence, developing new antibiotics will not be able to cope up the threats posed by the evolution of multidrug resistant strains of microorganisms. Hence, there is a vital and immediate need for the discovery of non-traditional medicines, against which microbes are unlikely to develop resistance. The new approach should be able to minimize the side-effects associated with the medication of traditional antibiotics. Metal and metal oxide based materials are widely used for the treatment of infectious diseases since ancient time. However, technological advancement in the field of antibiotics had declined interest in these conventional medicines in the 20th century. Recent developments of multidrug resistant strains of microorganisms have rejuvenated interest in the antimicrobial properties of metals and metal oxides. The major objective of this thesis is to synthesize metals (silver and copper) and metal oxide (titanium dioxide) nanoparticles and evaluate their antimicrobial properties against clinically important pathogens and environmental friendly microorganisms, which are critical to sustain natural cycles. First chapter of this thesis introduces subject of infection and infectious diseases. The details of epidemics caused by infectious diseases in the history of human civilization are overviewed to highlight the importance of this class of diseases. The concept of antibiotics is introduced, which is the only alternative available with pharmacologist for the treatment of infectious diseases. Detailed mechanism of microbial action of antibiotics is also reviewed along with their merits and demerits. The origin of resistance in microorganisms against antibiotics is reviewed in this introductory chapter. The need for alternate medical modality to fight against the threats raised by the development of resistance strains of microorganisms is pressed upon and the possibility of metals and metal oxide nanostructures to be used as non-conventional antibiotics to target multidrug resistance strains is explored. A brief review on possible environmental hazards of nanomaterial based non-traditional antibiotics is also presented in chapter - 1. The second half of chapter - 1 reviews the existing state of art of nanomaterials’ preparation with specific focus on tailored synthesis of metals (silver and copper) and metal oxide (titanium dioxide) nanostructures. Based on the existing literature, the correlation between processing parameters and their biological activities have been established. From the literature survey, it is found that no systematic correlation between the synthesis protocols and bioactivity of silver, copper and titanium dioxide nanoparticles exist, which can be used as authenticate and reliable source of information in nanomedicine. The second chapter includes description of various characterization tools/techniques, which are used to characterize nanomaterials synthesized to fulfill the objectives of this thesis. The detailed experimental protocols of each characterization technique (XRD, PCS, FTIR, UV-visible spectroscopy, ICP-AES, TG/DSC, TEM, SEM/EDS) along with sample preparation details and measurement conditions are presented. The third chapter is divided into three subparts. The first part of this chapter deals with the evolvement of synthesis protocols for size selective synthesis of silver nanoparticles. They are synthesized by chemical reduction technique in which oleylamine is used as both reducing and capping agent. Effect of various synthesis parameters like concentration of reducing/capping agent, nucleation temperature, nucleation time, growth temperature and growth time on nanoparticles’ yield, their hydrodynamic, crystallite and physical size, morphology and plasmonic characteristics are studied. From the detailed investigation of synthesized nanoparticles by various characterization techniques, the optimized conditions for high yield, nearly monodisperse, water dispersible silver nanoparticles that can be directly used as antimicrobial agent in nanomedicine are being laid down. Synthesis of copper nanoparticles by chemical reduction technique is described in the second part of chapter - 3. It has been established that three component system i.e. strong reducing agent (NaBH4), weak reducing agent (L-ascorbic acid) and surfactant (PVP) is essential for the formation of metallic copper phase. Uniform, nearly monodisperse, ultra-small copper nanoparticles obtained under ambient conditions are stable against aggregation and oxidation. The plausible mechanism responsible for stabilization of copper nanoparticles’ surface against oxidation is also provided. Third part of chapter - 3 describes synthesis of undoped and cobalt (0-2 wt%) doped Titanium dioxide nanoparticles by sol-gel technique. The effect of cobalt doping on the optical band gap of anatase phase of TiO2 nanoparticles have been evaluated by diffuse reflectance UV-visible spectroscopy. It has been found that increasing cobalt concentration in the TiO2 matrix reduces its band gap from 3.03 eV to 1.93 eV. By cobalt doping, the spectral response of TiO2 nanoparticles has been shifted from UV region to the visible region of the electromagnetic spectrum, which is an essential condition for photoinduced bioactivity of TiO2 nanoparticles. Evaluation of antimicrobial activities of as-synthesized and antibiotics adsorbed nanoparticles is the subject matter of chapter - 4. The antimicrobial activities of nanoparticles and antibiotic adsorbed nanoparticles have been evaluated against clinically important pathogenic strains of E. coli, S. aureus and P. vulgaris and eco-friendly microorganisms of B. subtilis and P. fluorescens. The antimicrobial activities of a particular antibiotic against pathogenic / eco-friendly microorganism under test have been evaluated in terms of minimum inhibitory concentration (MIC) and zone of inhibition (ZIH) by micro-dilution method and disk diffusion test, respectively. Highest biocidal activity is observed for copper nanoparticles amongst the studied nanoparticulate systems. The MIC values of copper nanoparticles ranges between 20-50 µg/mL. For silver nanoparticles, the MIC values lies in the range of 25-150 µg/mL. This observation is in contradiction with the existing literature where highest biocidal activity is reported for silver nanoparticles amongst all nano-antimicrobial systems. The exact reason behind observed enhanced antimicrobial activities of copper nanoparticles is not completely understood. This enhanced biocidal activity of copper nanoparticles could be ascribed to their colloidal stability, ultra-small size and high oxidation resistance. No significant inhibition of organism growth is observed under light or dark conditions in the tested concentration range (0 - 500 µg/mL) of TiO2. Irrespective of the optical band gap (i.e. Co-dopant concentration), no MIC of TiO2 is observed for pathogenic/ecofriendly microorganisms. The antimicrobial activities of silver nanoparticles increase by 60-346% for tetracycline adsorption and 70-289% for kanamycin adsorption on nanoparticles. For tetracycline adsorbed copper nanoparticles, the enhancement is in the range of 0-49% and for kanamycin adsorbed copper nanoparticles, it improves by 0-20%. From these results, it is concluded that when antibiotics adsorbed on silver nanoparticles, it drastically enhances the biocidal activities of silver nanoparticles while same is not happening for copper nanoparticles. This might be because of adsorption of copper nanoparticles at biologically active sites of antibiotics. The phenomenological description of mechanism governing the microbial activities of nanoparticles is also provided in this chapter. Chapter - 5 summarizes the important findings along with the scope for future work in the proposed field of antibacterial nanobiotechnology.