Nanoparticle mediated toxicity and antimicrobial action

Nanomaterials are either inorganic or organic nanosized particles which have many industrial and biological applications such as in cosmetics, environmental remediation, electronics, biosensing and imaging and in drug delivery. Some have toxic effect upon release to the environment causing death of microorganisms and others are biodegradable and nontoxic to the living beings. In this work, two types of nanoparticles were investigated: inorganic titania nanoparticles which have been shown to have toxic effects and organic Carbopol Aqua SF1 microgel particles which were shown to be nontoxic and biodegradable organic nanoparticles for drug delivery.Chapter one explores the current literature relating to nanoparticles. The types, chemical and physical properties, methods of synthesis, characterization and functionalization are discussed along with general applications and the toxicity of titania nanoparticles. The role of nanomaterials as drug delivery systems and their design in terms of stability, swelling studies, encapsulation, drug loading and release, response to stimuli and targeting is also discussed. Finally the use of microfluidics for screening nanoparticle activity is discussed including microfabrications of chips cell trapping methods and microfluidic cell based assay methods.Chapter two is the experimental chapter describing the chemicals and instrumentation used. It also includes the methods used for the synthesis of titania nanoparticles and effects of pH on the zeta potential measurement. In addition to that, methods for the testing of the cytotoxic effects of uncoated and coated titania nanoparticles are described. Finally the methods employed for studying the optimization of the encapsulation of berberine and chlorhexidine into Carbopol Aqua SF1 are also included.Chapter three describe the synthesis of titania nanoparticles (TiO2NPs) and their characterization, including crystallite size, particle size distribution, surface area measurement and zeta potential. It was found that as the temperature increased from 100oC to 800oC, the crystallite size and particle size increased while the surface area decreased. At 100oC, the crystallite size, particle size, surface area and zeta potential of the titania nanoparticles were 5 nm, 25±20 nm, 163 m2 g-1 and +40±9 mV, respectively with anatase as the dominant phase. However, the phase changed to rutile at the annealing temperature of 800oC with the crystallite size, particle size, surface area and zeta potential becoming 142 nm, 145±60 nm, 7.5 m2 g-1 and -26±8 mVrespectively. In addition to that, the zeta potential of the titania nanoparticles at 25 nm size was affected by changing the pH of the suspension, at low pH, the zeta potential was +40 mV giving high stability and fully dispersed particles while the nanoparticles flocculated in the basic medium with a zeta potential of -25 mV, with the isoelectric point of titania nanoparticles being pH 6.7. Changing the pH of the solution for titania nanoparticles caused an irreversible process as it was not possibility to convert the aggregated titania nanoparticles from microscale to nanoscale.Chapter four describes the investigation into the the nanotoxicity of the titania nanoparticles (TiO2NPs) at various hydrodynamic diameters and crystallite size on C. reinhardtii microalgae and S. cerevisiae (yeast) upon illumination with UV and visible light. The cell viability was assessed for a range of nanoparticle concentrations and incubation times. It was found that uncoated TiO2NPs affect the C. reinhardtii cell viability at a much lower particle concentrations than for yeast. It was also observed that the TiO2NPs toxicity increased upon illumination with UV light compared to dark conditions due to the oxidative stress of the reactive oxygen species produced. It was also found that TiO2NPs nanotoxicity increased upon illumination with visible light which indicated that the nanoparticles might also interfere with the microalgae photosynthetic system leading to decreased chlorophyll content upon exposure to TiO2NPs. The results showed that the larger the hydrodynamic diameter of the TiO2NPs the lower their nanotoxicity, with anatase TiO2NPs generally being more toxic than rutile TiO2NPs. A range of polyelectrolyte-coated TiO2NPs were also prepared using the layer by-layer method and their nanotoxicity on yeast and microalgae was studied. It was found that the toxicity of the coated TiO2NPs alternates with their surface charge. TiO2NPs coated with cationic polyeletrolyte as an outer layer exhibited much higher nanotoxicity than the ones with an outer layer of anionic polyelectrolyte. TEM images of sectioned microalgae and yeast cells exposed to different polyelectrolyte-coated TiO2NPs confirmed the formation of a significant build-up of nanoparticles on the cell surface for bare and cationic polyelectrolyte-coated TiO2NPs. The effect came from the increased adhesion of cationic nanoparticles to the cell walls. Significantly, coating the TiO2NPs with anionic polyelectrolyte as an outer layer led to a reduced adhesion and much lower nanotoxicity due to electrostatic repulsion with the cell walls.Chapter five describes the development and characterisation of berberine-loaded and chlorhexidine-loaded polyacrylic acid based microgels. The procedure for loading the Carbopol microgels with both berberine and chlorhexidine was developed using a swelling-deswelling cycle dependent on pH. The result of this protocol was a colloidal suspension of collapsed microgel particles loaded with fixed percentage of the antimicrobial agents, berberine and chlorhexidine, respectively. The initial microgel particle concentration, as well as the initial concentrations of berberine and chlorhexidine, were optimized to allow for maximum encapsulation efficiency of the loaded reagent in the microgel while maintaining the colloidal stability of the Carbopol microgel suspension. It was determined that 0.15 wt% berberine and 0.1% chlorhexidine could be successfully incubated with 0.1 wt% Carbopol microgel while the pH was varied from 8 to 5.5 with a measurable increase of the collapsed microgel due to electrostatic conjugation of these cationic antimicrobial agents with the carboxylic groups of the microgel.While for berberine, only 10% encapsulation efficiency was achieved, for chlorhexidine over 90% encapsulation efficiency was obtained without significant impact on the colloidal stability of the microgel. The zeta potential of the loaded microgels remained negative in the range of -35 mV - -40 mV with very moderate increase of the collapsed (and loaded) microgel particle size. The release of berberine and chlorhexidine from these microgel materials was studied and sustained release from the formulations was demonstrated upon dilution over the period of up to 6 hours. The berberine- and chlorhexidine-loaded microgel particles were then further coated with cationic polyelectrolytes, PAH and PDAC. This carried out to increase the adhesion of these antimicrobial particles to the cell membranes. These studies showed a reversal of the zeta-potential of the PDAC coated microgels after their loading with berberine and chlorhexidine, respectively.In chapter six, the antimicrobial activity of both berberine and chlorhexidine loaded Carbopol microgel was studied upon incubation with algae, yeast and E.coli. It was noticed that an increase in the antimicrobial activity of berberine and chlorhexidine Carbopol microgel occurred after 6 hours incubation time for algae and after 24 hours for E.coli while there was no pronounced antimicrobial action for yeast in comparison with the antimicrobial activity of free berberine or chlorhexidine. This was due to the repulsion forces between the anionic microgel and the anionic cell membrane which did not allow the encapsulated berberine or chlorhexidine to be released and diffuse into the cytoplasm causing cell death. In addition to that, the fully anionic charged Carbopol microgel did not allow berberine and chlorhexidine to be released easily at pH 5.5 while the percentage of release increased with pH up to 7.5.The antimicrobial activity of cationic PDAC coated berberine and chlorhexidine loaded-Carbopol microgel was also studied for algae, yeast and E.coli. It was found that cationic PDAC on its own had an acute toxic effect on algae, yeast and E.coli while the toxicity of cationic PDAC reduced upon coating Carbopol microgel with cationic PDAC. Algae and E.coli stayed viable up to 0.0045 wt. % and 0.009 wt. % of PDAC coated Carbopol microgel, respectively. Yeast was resistance to the PDAC coated carbopol for a wide range of concentrations of PDAC coated Carbopol microgel up to 0.018 wt. %. This was due to the different thicknesses of the cell membrane. The Carbopol microgels with encapsulated berberine and chlorhexidine were then coated with cationic PDAC to form PDAC coated particles. The PDAC coated berberine or chlorhexidine loaded carbopol microgel were then incubated with each of algae, yeast, and E.coli. The coating appeared to increase the antimicrobial actions against algae, yeast and E.coli for short incubation times. The increase in the antimicrobial activity was attributed to the electrostatic interaction between the cationic PDAC coated berberine or chlorhexidine loaded carbopol microgel and the anionic cell membrane allowing diffusion of berberine or chlorhexidine easily through cell membrane causing cell death. TEM images showed aggregation of these cationic PDAC coated berberine or chlorhexidine loaded carbopol microgel on the surface of the cell membrane.Chapter seven describes the development of new microfluidics device for cell trapping to achieve a microscreening cell based assay. The idea involved trapping the cells in a micro chamber and then passing over suspensions of the nanomaterials and monitoring the effect. Three design of microfluidic chips were studied with different designs, channel dimensions (depth and width) cell trapping techniques and materials. Initially chemical adhesion was investigated to adhere cells into micro chamber of the microfluidics device using poly-l-lysine bu the cells detached from the surface of microchip because of shear stress forces. Synthesized magnetic yeast cells which were then investigated for trapping other cells into the micro chamber of the chip but the back pressures were too high when liquids were flowed through the system. Magnetic glass beads were then studied to trap the cells, these were synthesized by coating anionic glass beads with cationic and anionic polyelectrolytes such as PAH and PSS, however, they had a low magnetic response towards the magnet. Synthesized magnetic beads were then synthesized using a PDMS based ferrofluid but again a low magnetic response was obtained towards the magnet. Synthesis by flow focusing microfluidics was the tried to generate mono dispersed magnetic beads where SDS with water was the continuous phase and a styrene based ferrofluid was the dispersed phase but the magnetic beads were unstable and they need to be optimized. The continuous phase was then changed to use Hitenol BC20 a polymerisable surfactant, to form hydrophobic magnetic beads and and a mixing serpentine was added to the chip design to give the generated magnetic beads time to be stablilise. Despite these changes the magnetic beads stayed unstable and therefore an emulsification method was used to fabricate poly dispersed magnetic beads which produced 20μm to 50μm magnetic beads. These beads were successfully utilized for trapping cells into the micro chamber of the chip device. A new microfluidic device was designed with suitable channel dimensions which allowed the magnetic beads to move freely inside the micro chamber of the device. These beads were placed into the micro chamber could be controlled using the neodymium magnet easily move the beads

Nanotoxicity of polyelectrolyte-functionalized titania nanoparticles towards microalgae and yeast: Role of the particle concentration, size and surface charge

We studied the nanotoxicity of titania nanoparticles (TiO₂NPs) of various hydrodynamic diameters and crystallite sizes towards C. reinhardtii microalgae and S. cerevisiae (yeast) upon illumination with UV and visible light. The cell viability was assessed for a range of nanoparticle concentrations and incubation times. We found that bare TiO₂NPs affect the C. reinhardtii cell viability at much lower particle concentrations than for yeast. We observed an increase of the TiO₂NPs toxicity upon illumination with UV light compared with that in dark conditions due to the oxidative stress of the produced reactive oxygen species. We also found an increased TiO₂NPs nanotoxicity upon illumination with visible light which indicates that they may also interfere with the microalgae's photosynthetic system leading to decreased chlorophyll content upon exposure to TiO₂NPs. The results indicate that the larger the hydrodynamic diameter of the TiO₂NPs the lower is their nanotoxicity, with anatase TiO₂NPs generally being more toxic than rutile TiO₂NPs. We also prepared a range of polyelectrolyte-coated TiO₂NPs using a layer by-layer method and studied their nanotoxicity towards yeast and microalgae. We found that the toxicity of the coated TiO₂NPs changes with their surface charge. TiO₂NPs coated with cationic polyelectrolyte as an outer layer exhibit much higher nanotoxicity than the ones with an outer layer of anionic polyelectrolyte. TEM images of sectioned microalgae and yeast cells exposed to different polyelectrolyte-coated TiO₂NPs confirmed the formation of a significant build-up of nanoparticles on the cell surface for bare and cationic polyelectrolyte-coated TiO₂NPs. The effect comes from the increased adhesion of cationic nanoparticles to the cell walls. Significantly, coating the TiO₂NPs with anionic polyelectrolyte as an outer layer led to a reduced adhesion and much lower nanotoxicity due to electrostatic repulsion with the cell walls. This suggest a new way of making cationic TiO₂NPs safer for use in different formulations by pre-coating them with anionic polyelectrolytes. The results of this study give important insights into the various factors controlling the nanotoxicity of TiO₂NPs

Controlling the nanotoxicity of polyelectrolyte-functionalized titania nanoparticles

This study gives important insights of the various factors controlling the nanotoxicity of titania nanoparticles (TiO2NPs). We studied the nanotoxicity of TiO2NPs of various hydrodynamic diameters and crystallite sizes on C. Reinhardtii (microalgae) and S. cerevisiae (yeast) upon illumination with UV/visible light [1]. The cell viability was assessed for a range of nanoparticle concentrations and incubation times. Bare TiO2NPs affect the microalgae viability at much lower particle concentrations than for yeast. We also found an increased nanotoxicity upon illumination with visible light which indicates that they may also interfere with the microalgae photosynthetic system leading to decreased chlorophyll content upon exposure to TiO2NPs. The results indicate that the larger the hydrodynamic diameter of the TiO2NPs the lower is their nanotoxicity, with anatase TiO2NPs generally being more cytotoxic than rutile TiO2NPs. We also prepared a range of polyelectrolyte-coated TiO2NPs using the layer by-layer method and studied their nanotoxicity on yeast and microalgae. The toxicity of the coated TiO2NPs alternates with their surface charge. TiO2NPs coated with cationic polyelectrolyte as an outer layer exhibit much higher nanotoxicity than the ones with an outer layer of anionic polyelectrolyte. TEM images of sectioned microalgae and yeast cells exposed to different polyelectrolyte-coated TiO2NPs confirmed the formation of a significant build-up of nanoparticles on the cell surface for bare- and cationic polyelectrolyte-coated TiO2NPs. The effect is coming from the increased adhesion of cationic nanoparticles to the cell walls. Significantly, coating the TiO2NPs with an anionic polyelectrolyte as an outer layer led to a reduced adhesion and much lower nanotoxicity due to electrostatic repulsion with the cell walls. This suggest a new way of making the TiO2NPs potentially safer for use in different formulations by pre-coating them with anionic polyelectrolytes. Please click Additional Files below to see the full abstract

Enhanced antimicrobial effect of berberine in nanogel carriers with cationic surface functionality

We report a strong enhancement in the antimicrobial action of berberine encapsulated into polyacrylic acid-based nanogels followed by further surface functionalisation with a cationic polyelectrolyte (PDAC). Due to the highly developed surface area, the nanogel carrier amplifies the contact of berberine with microbial cells and increases its antimicrobial efficiency. We show that such cationic nanogel carriers of berberine can adhere directly to the cell membranes and maintain a very high concentration of berberine directly on the cell surface. We demonstrated that the antimicrobial action of the PDAC-coated nanogel loaded with berberine on E. coli and C. reinhardtii is much higher than that of the equivalent solution of free berberine due to the electrostatic adhesion between the positively charged nanogel particles and the cell membranes. Our results also showed a marked increase in their antimicrobial action at shorter incubation times compared to the non-coated nanogel particles loaded with berberine under the same conditions. We attribute this boost in the antimicrobial effect of these cationic nanocarriers to their accumulation on the cell membranes which sustains a high concentration of released berberine causing cell death within much shorter incubation times. This study can provide a blueprint for boosting the action of other cationic antimicrobial agents by encapsulating them into nanogel carriers functionalised with a cationic surface layer. This nanotechnology-based approach could lead to the development of more effective wound dressings, disinfecting agents, antimicrobial surfaces, and antiseptic and antialgal/antibiofouling formulations

Toxicity of polyelectrolyte-functionalized titania nanoparticles in zebrafish (Danio rerio) embryos

We investigated the effects of short-term exposure of bare TiO2NPs and polyelectrolyte-coated TiO2NPs in the 5-25 nm size range, at relatively high concentrations (of 500 and 1000 mg/L) under light or dark conditions, in D. rerio embryos. The biological endpoints investigated included embryo viability and mRNA transcript levels of antioxidant and membrane transport genes relative to control embryos. The presence of nanoparticles on the surface of embryos was assessed using TEM. The results confirm an accumulation of TiO2NPs on the outer surface (chorion) of the embryo, but not within the embryo. No significant difference in embryo viability was detected following each exposure regime. The expression of antioxidant biomarker, SOD2, was significantly impacted by the type of TiO2NP, with TiO2NPs/PSS/PAH coating exposure showing down regulation; the concentration of the nanoparticles, with down regulation at 500 mg/L; and dark/light condition with down regulation in the light. The expression levels of the hypoxia and membrane markers, HIF1 and Pxmp2, were not significantly impacted by any factor. The study indicates that SOD2 mRNA expression levels may be useful in the detection of apparent oxidative stress induced by the titania nanoparticle build up on the embryo chorion surface

Design, synthesis, molecular modeling and biological evaluation of novel diaryl heterocyclic analogs as potential selective cyclooxygenase-2 (COX-2) inhibitors

AbstractNew series of 3,4-diaryl-2-thioxoimidazolidin-4-ones and 3-alkylthio-4,5-diaryl-4H-1,2,4-triazoles were designed, synthesized and evaluated for their activity as anti-inflammatory agents. Compounds 20, 21, 23 and 34 are highly selective inhibitors of COX-2 enzyme at a concentration of 100mM relative to celecoxib, the standard reference. (±)-3-(4-Phenoxy-phenyl)-5-phenyl-2-thioxoimidazolidin-4-ones 23 exhibited the most active anti-inflammatory agent

Amplified antimicrobial action of chlorhexidine encapsulated in PDAC-functionalized acrylate copolymer nanogel carriers

We have developed and tested a novel functionalised nanocarrier for chlorhexidine (CHX) which provides a very strong enhancement of its antimicrobial action. The nanocarrier was based on lightly-cross-linked acrylate copolymer nanogel particles loaded with CHX followed by a surface functionalisation with the cationic polyelectrolyte poly(diallyldimethylammonium chloride (PDAC). We explored the antimicrobial effect of the PDAC-coated CHX-loaded nanogel carriers on E. coli, S. auresus, C. cerevisiae and C. reinhardtii and discovered that it is much higher than that of solution with equivalent overall concentration of free CHX. Our experiments also showed a marked increase of the cationic CHX-loaded nanocarriers antimicrobial action on these microorganisms at shorter incubation times compared with the non-coated CHX-loaded nanogel particles at the same CHX concentration and other conditions. We attribute the increase in the antimicrobial activity of the cationically-functionalised nanogel carrier to its electrostatic adhesion to the microbial cells walls which allows much higher CHX concentration to be delivered directly onto the cell surface. The results of this study can be used for development of novel more efficient antialgal, antifungal and antbacterial formulations based on cationically functionalised nanogels. Our method can also be used for boosting the effect of other cationic antimicrobial agents by encapsulating them in cationically-functionalised nanogel carriers. This nanotechnological approach could lead to developing more effective antimicrobial and disinfecting agents, dental formulations for plaque control, wound dressings, antialgal/antibiofouling formulations and novel antifungal agents

Harnessing genomics to improve health in the Eastern Mediterranean Region – an executive course in genomics policy

BACKGROUND: While innovations in medicine, science and technology have resulted in improved health and quality of life for many people, the benefits of modern medicine continue to elude millions of people in many parts of the world. To assess the potential of genomics to address health needs in EMR, the World Health Organization's Eastern Mediterranean Regional Office and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20(th)–23(rd), 2003, in Muscat, Oman. The 4-day course was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health. The overall objective of the course was to collectively explore how to best harness genomics to improve health in the region. This article presents the course findings and recommendations for genomics policy in EMR. METHODS: The course brought together senior representatives from academia, biotechnology companies, regulatory bodies, media, voluntary, and legal organizations to engage in discussion. Topics covered included scientific advances in genomics, followed by innovations in business models, public sector perspectives, ethics, legal issues and national innovation systems. RESULTS: A set of recommendations, summarized below, was formulated for the Regional Office, the Member States and for individuals. • Advocacy for genomics and biotechnology for political leadership; • Networking between member states to share information, expertise, training, and regional cooperation in biotechnology; coordination of national surveys for assessment of health biotechnology innovation systems, science capacity, government policies, legislation and regulations, intellectual property policies, private sector activity; • Creation in each member country of an effective National Body on genomics, biotechnology and health to: - formulate national biotechnology strategies - raise biotechnology awareness - encourage teaching and training of biotechnology - devise integration of biotechnology within national health systems. CONCLUSION: The recommendations provide the basis for a road map for EMR to take steps to harness biotechnology for better and more equitable health. As a result of these recommendations, health ministers from the region, at the 50th Regional Committee Meeting held in October 2003, have urged Member States to establish national bodies of biotechnology to formulate a strategic vision for developing biotechnology in the service of the region's health. These efforts promise to raise the profile of genomics in EMR and increase regional cooperation in this exciting new field

Investigation of Anti-MRSA and Anticancer Activity of Eco-Friendly Synthesized Silver Nanoparticles from Palm Dates Extract

In the present work, we investigated novel, green, and facile approach for the synthesis of stable silver nanoparticles by use of inexpensive and available Iraqi Zahdi palm dates extract as reducing and stabilizing agents. The formation and color change of dark brown suspension of silver nanoparticles were monitored and examined by ultraviolet-visible spectrophotometric analysis at maximum wavelength of 401 nm. The produced nanoparticles were characterized by dynamic light scattering (DLS), Fourier transform-infrared spectrometry (FTIR), atomic force microscope (AFM), X-ray diffraction (XRD) and scanning electron microscopy (SEM)-energy dispersive spectroscopy (EDS). The results demonstrated that particle size distribution was around 90 ± 40 nm which was in line with grain size of the nanoparticles measured by AFM, while the crystallite size was about 20 nm according to the results observed from XRD technique. Additionally, the zeta potential of synthesized silver nanoparticles was found to be nearly about -25 mV, whereby the suspension showed high stability upon storing the sample in a cool place for a long period of time. The synthesized silver nanoparticles exhibited efficient antibacterial activity against pathogenic methicillin-resistant Staphylococcus aureus (MRSA) bacteria. They also showed enhanced anticancer activity against breast cancer cells (MCF7) with high apoptotic effect as compared with normal MCF10A cells, which confirmed the biocompatible nature of green synthesized silver nanoparticles. It is considered that nanosilver particles synthesized with palm dates extract may basically establish a significant step to synthesize such nanoparticles for topical applications as well as an alternative anticancer drug of existing chemotherapeutics of cancer.

Silver Nanoparticles as an Effective Anti-Nanobacterial System towards Biofilm Forming Pseudomonas oryzihabitans

Silver nanoparticles have been considered a powerful antimicrobial agents recently especially after increasing incidence of diseases associated with biofilm and multi-drug resistant pathogens required to find a novel path to eradicate that challenge. The present study aims to evaluate the antibacterial activity of biosynthesized silver nanoparticles (AgNPs) using a cell-free extract of Enterobacter cloacae and chemo synthesis by sodium borohydride (NaBH4) on biofilm-forming Pseudomonas oryzihabitans. Antimicrobial effect of silver nanoparticles in both types and in combination with imipenem were evaluated by agar well diffusion method. The results revealed a good response to inhibit biofilm-forming Pseudomonas oryzihabitans growth by silver nanoparticles antibacterial activity in both types (biological and chemical) and in combination with imipenem; the antimicrobial effect was increased and enhanced. In the present study, it was found that the biological and chemical silver nanoparticles were considered a novel and decisive solution against biofilm and multi- drug resistance bacteria with a preference of biological silver nanoparticles