Nanoparticles for Signaling in Biodiagnosis and Treatment of Infectious Diseases

[EN]Advances in nanoparticle-based systems constitute a promising research area with important implications for the treatment of bacterial infections, especially against multidrug resistant strains and bacterial biofilms. Nanosystems may be useful for the diagnosis and treatment of viral and fungal infections. Commercial diagnostic tests based on nanosystems are currently available. Different methodologies based on nanoparticles (NPs) have been developed to detect specific agents or to distinguish between Gram-positive and Gram-negative microorganisms. Also, biosensors based on nanoparticles have been applied in viral detection to improve available analytical techniques. Several point-of-care (POC) assays have been proposed that can offer results faster, easier and at lower cost than conventional techniques and can even be used in remote regions for viral diagnosis. Nanoparticles functionalized with specific molecules may modulate pharmacokinetic targeting recognition and increase anti-infective efficacy. Quorum sensing is a stimuli-response chemical communication process correlated with population density that bacteria use to regulate biofilm formation

Investigation of solution-phase and on-chip binding of C-reactive proteins and antibodies

Gold nanoparticles can self-assemble into nanostructures in the presence of suitable linker molecules. The self-assembly of gold nanoparticles functionalized with C-reactive protein (CRP) antibodies in the presence of CRP antigen linker molecules was explored. A ratio of antigen linker molecules to nanoparticle (2:1) that resulted in rapid nanoparticle self- assembly was identified, evidenced as a distinct solution colour change from red to blue within 5 minutes. Higher linker molecule- nanoparticle ratios (12:1, 18:1, 72:1) resulted in slow formation of nanostructures and only a slight solution colour change (even after periods of several days), the rate being dependent on the number of available binding sites. The propensity of nanoparticles to rapidly assemble into nanostructures at certain linker molecule- nanoparticle ratios was corroborated employing citrate-stabilized nanoparticles and di- isothiocyanate terminated metal-organic rhenium linker molecules, whereby again rapid formation of nanostructures was dependent on specific molecule-nanoparticle ratios as distinct from other molecule-nanoparticle ratios. UV-visible spectroscopy and scanning electron microscopy characterization confirmed visual observations. Surface-based assays also show much promise in application to point-of-care detection. It was of interest to determine if surface-based assays void of complex chemical processes and elaborate equipment could compete with laboratory-based assays in terms of specificity, stability and sensitivity but also offer faster and inexpensive diagnosis. The binding of CRP antibody to silanised silicon-silicon oxide substrates implemented using the organosilane APTES and the subsequent binding of CRP antigen to this immobilised CRP antibody was explored. The binding event of CRP antigen to surface immobilised CRP antibody was electrically characterised in ambient conditions. The formation of the antibody-antigen complex resulted in a corresponding decrease of the sensor capacitance at a CRP antigen concentration of 20 µg/mL (2 hours). However, it was demonstrated that nanoparticle amplification can facilitate rapid visual surface-based detection of CRP antigen of between 5 and 20 µg/mL

Development Of Molecular Biosensors And Multifunctional Graphene-Based Nanomaterials

In the first project, a simple, rapid, and reversible fluorescent DNA INHIBIT logic gate has been developed for sensing mercury (Hg2+) and iodide (I-) ions based on a molecular beacon (MB). In this logic gate, a mercury ion was introduced as the first input into the MB logic gate system to assist in the hybridization of the MB with an assistant DNA probe through the thymine–Hg2+–thymine interaction, which eventually restored the fluorescence of MB as the output. With this signal-on process, mercury ions can be detected with a limit of detection as low as 7.9 nM. Furthermore, when iodide ions were added to the Hg2+/MB system as the second input, the fluorescence intensity decreased because Hg2+ in the thymine–Hg2+–thymine complex was grabbed by I- due to a stronger binding force. Iodide ions can be detected with a limit of detection of 42 nM. Meanwhile, we studied the feasibility and basic performance of the DNA INHIBIT logic gate, optimized the logic gate conditions, and investigated its sensitivity and selectivity. The results showed that the MB based logic gate is highly selective and sensitive for the detection of Hg2+ and I- over other interfering cations and anions. In the second project, an ultrasensitive and rapid turn-on fluorescence assay has been developed for the detection of 3’-5’ exonuclease activity of exonuclease III (Exo III) using molecular beacons (MBs). This method has a linear detection range from 0.04 to 8.00 U mL-1 with a limit of detection of 0.01 U mL-1. In order to improve the selectivity of the method, a dual-MB system has been developed to distinguish between different exonucleases. With the introduction of two differently designed MBs which respond to different exonucleases, the T5 exonuclease, Exo III and RecJf exonucleases can be easily distinguished from each other. Furthermore, fetal bovine serum and fresh mouse serum were used as complex samples to investigate the feasibility of the dual-MB system for the detection of the enzymatic activity of Exo III. As a result, the dual-MB system showed a similar calibration curve for the detection of Exo III as in the ideal buffer solution. The designed MB probe could be a potential sensor for the detection of Exo III in biological samples. In the third project, A sensitive label-free fluorescence assay for monitoring T4 polynucleotide kinase (T4 PNK) activity and inhibition was developed based on a coupled λ exonuclease cleavage reaction and SYBR Green I. In this assay, a double-stranded DNA (dsDNA) was stained with SYBR Green I and used as a substrate for T4 PNK. After the 5´-hydroxyl termini of the dsDNA was phosphorylated by the T4 PNK, the coupled λ exonuclease began to digest the dsDNA to form mononucletides and single-stranded DNA (ssDNA). At this moment, the fluorescence intensity of the SYBR Green I decreased because less affinity with ssDNA than dsDNA. The decrease extent was proportional to the concentration of the T4 PNK. After optimization of the detection conditions, including the concentration of ATP, amount of λ exonuclease and reaction time, the activity of T4 PNK was monitored by the fluorescence measurement, with the limit of detection of 0.11 U/mL and good linear correlation between 0.25-1.00 U/mL (R2=0.9896). In this assay, no label was needed for the fluorescence detection. Moreover, the inhibition behaviours of the T4 PNK’s inhibitors were evaluated by this assay. The result indicated a potential of using this assay for monitoring of phosphorylation-related process. In the fourth project, a facile bottom-up method for the synthesis of highly fluorescent graphene quantum dots (GQDs) has been developed using a one-step pyrolysis of a natural amino acid, L-glutamic acid, with the assistance of a simple heating mantle device. The developed GQDs showed strong blue, green and red luminescence under irradiation with ultra-violet, blue and green light, respectively. Moreover, the GQDs emitted near-infrared (NIR) fluorescence in the range 800–850 nm with an excitation-dependent manner. This NIR fluorescence has a large Stokes shift of 455 nm, providing a significant advantage for the sensitive determination and imaging of biological targets. The fluorescence properties of the GQDs, such as the quantum yields, fluorescence life times, and photostability, were measured and the fluorescence quantum yield was as high as 54.5%. The morphology and composites of the GQDs were characterized using TEM, SEM, EDS, and FT-IR. The feasibility of using the GQDs as a fluorescent biomarker was investigated through in vitro and in vivo fluorescence imaging. The results showed that the GQDs could be a promising candidate for bioimaging. Most importantly, compared to the traditional quantum dots (QDs), the GQDs are chemically inert. Thus, the potential toxicity of the intrinsic heavy metal in the traditional QDs would not be a concern for GQDs. In addition, the GQDs possessed an intrinsic peroxidase-like catalytic activity that was similar to graphene sheets and carbon nanotubes. Coupled with 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), the GQDs can be used for the sensitive detection of hydrogen peroxide with a limit of detection of 20 mM. In the fifth project, a general, environmental-friendly, one-pot method for the fabrication of reduced graphene oxide (RGO)/metal (oxide) (e.g. RGO/Au, RGO/Cu2O, and RGO/Ag) nanocomposties was developed using glucose as the reducing agent and stabilizer. The RGO/metal (oxide) nanocomposites were characterized using STEM, FE-SEM, EDS, UV-vis absorption spectroscopy, XRD, FT-IR and Raman spectroscopy. The reducing agent, glucose, not only reduced GO effectively to RGO, but it also reduced the metal precursors to form metal (oxide) nanoparticles on the surface of RGO. Moreover, the RGO/metal (oxide) nanocomposites were stabilized by gluconic acid on the surface of RGO. Finally, the developed nanomaterials were successfully applied to simultaneous electrochemical analysis of L-ascorbic acid (L-AA), dopamine (DA) and uric acid sing RGO/Au nanocomposite as an electrode catalyst. In the sixth project, a reduced graphene oxide/silver nanoparticle (RGO/Ag) nanocomposite using glucose as the environmental-friendly reducing agent was developed. The antibacterial activity of RGO/Ag nanocomposite was carefully investigated using Escherichia coli (E. coli) and Klebsiella pneumoniae (Kp) as bacterial models. We found that, compared with AgNPs, graphene oxide (GO) and RGO, RGO/Ag nanocomposite had higher antibacterial efficiency. Furthermore, under the near-infrared (NIR) irradiation, RGO/Ag nanocomposite demonstrated enhanced synergetic antibacterial activity through the photothermal effect. Almost 100 % of E. coli and Kp were killed by the treatment of 15 µg/mL and 20 µg/mL, respectively, with NIR irradiation. Moreover, the membrane integrity assay and ROS species assay demonstrated that RGO/Ag nanocomposite under NIR irradiation caused the cell membranes disruption and generation of ROS species, providing other possible mechanisms for their high antibacterial activity besides photothermal effect. In the seventh project, a rigid distance spacer, silica shell, was used between GO and dyes in this work to elucidate the quenching ability of GO. First, an organic dye was doped in silica nanoparticles, followed by the modification of another layer of silica shell with a different thickness. Due to the electrostatic interaction between GO and positively charged silica nanoparticles, GO wrapped the silica nanoparticles when they were mixed together. Therefore, the distance between GO and organic dyes was adjusted by the thickness of the silica shell. The quenching efficiency of GO to two different organic dyes, including Tetramethylrhodamine (TAMRA) and Tris(bipyridine)ruthenium(II) chloride (Rubpy), was measured at various distances. This quenching ability investigation of GO to dyes with distance-dependent manner would provide a guideline for the design of the fluorescent functional composite using GO in the future. In the eighth project, we characterized the antibacterial activity of GO in both cell culture and animal models. Klebsiella pneumoniae (Kp) is one of the most common multidrug resistant (MDR) pathogens in causing persistent nosocomial infections and is very difficult to eradicate once established in the host. First, we demonstrated that GO exerted direct killing of Kp in agar dishes and afforded the protection of alveolar macrophages (AM) from Kp infection in culture. We then evaluated the mortality, tissue damage, polymorphonuclear neutrophil (PMN) penetration, and bacterial dissemination in Kp-infected mice. Our results revealed that GO can counteract the invasive ability of Kp in vivo, resulting in lessened tissue injury, significant but subdued inflammatory response, and prolonged mouse survival. These findings indicate that GO may be an alternative agent for controlling MDR pathogens in clinics

Nano-engineered materials to probe and affect natural killer cell function

The nanoscale biological structures and dynamics that occur within cells have slowly been recognized as important features that determine cellular function. In the field of Immunology the receptors which determine a cell's response have recently been observed to organize on the nanoscale, forming clusters which vary in size, shape, and density. While these supramolecular structures have been observed, their function is still unknown, and current methods used to discern their function could be improved. Here, biomaterials with spatially controlled nanoscale features have been produced and developed with this goal in mind. Engineered nanomaterials have advantages over many biological techniques, especially in nanoscale manipulation and reduced experimental complexity. In this thesis two new nanoscale biomaterial approaches are developed, focusing on controlling the nanoscale spatial presentation of ligands to Natural Killer (NK) cells. The first is the fabrication of a supramolecular reagent which clusters Monoclonal Antibodies (mAbs) using a Nanoscale Graphene Oxide (NGO) framework. These clusters are on the same scale and density as receptor nanoclusters and exhibit properties that better mimic natural receptor ligation---such as interconnected ligands and local ligand density. Nanoscale Graphene Oxide-Monoclonal Antibody (NGO-mAb) species were found to augment degranulation and cytokine secretion of primary (human) NK (pNK) cells in comparison to equivalent concentrations of unclustered mAbs. Secondly, gold nanoparticle arrays, a nanotechnology used to create mimetic cell surfaces with nanometer control of ligand spacing, were imaged with Stochastic Optical Reconstruction Microscopy (STORM), a single molecule localization technique, to characterize their biological functionalization. This characterization step is necessary to confirm the nanoscale patterning, but remains difficult, and new methods for its accurate measurement are needed. Results obtained indicate that in spite of measured underoccupation, regions could be discerned as having spatial properties and patterning that would be expected of a functionalized array by three independent analytical methods.Open Acces

Utilizing the K18-hACE2 mouse model to develop protective COVID-19 vaccines

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic is caused by the respiratory virus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Similar to other respiratory viruses, SARS-CoV-2 is transmitted through inhalation of respiratory droplets and aerosols from infected individuals. Once inhaled, SARS-CoV-2 utilizes the receptor binding domain (RBD) on the spike protein to bind to human Angiotensin Converting Enzyme 2 (hACE2) receptor to gain entrance into host cells to begin viral replication. SARS-CoV-2 infection can result in mild to severe cases of COVID-19 ranging from asymptomatic infections, cold or flu like symptoms to respiratory failure. The onset of the pandemic in 2019 triggered a push to develop vaccines and therapeutics to prevent and treat SARS-CoV-2 infections. At the end of 2020, companies such as Moderna and Pfizer began to administer the first COVID-19 mRNA vaccines, and now in 2022, there are ten World Health Organization (WHO) approved vaccines with many more vaccines in clinical trials and pre-clinical development. At this time, approximately 12 billion doses have been administered worldwide accounting for 61% of the global population being fully vaccinated. However, with the continual emergence of SARS-CoV-2 variants of concern (VOC), each harboring new mutations that can negatively impact vaccine efficacy, there is a need to study and develop new vaccine approaches to improve immunity against VOC. Here, we devised three approaches to help improve vaccine efficacy against SARS-CoV-2 using the pre-clinical Keratin promoter 18-human Angiotensin Converting Enzyme 2 (K18-hACE2) mouse model. First, we evaluated the pathogenesis and response of VOC against human convalescent plasma (HCP) obtained from patients infected with the ancestral strain of SARS-CoV-2. Second, we assessed the vaccine efficacy of four adjuvanted Beta VOC or ancestral strain derived RBD Virus-like particle (VLP) vaccines against Alpha and Beta VOC challenge. Third, we evaluated intranasal administration of a RBD carrier protein-based vaccine adjuvanted with a lipid A mimetic. K18-hACE2 challenge models were used to establish SARS-CoV-2 VOC lethal challenge doses for Alpha, Beta, and Delta. Once a lethal viral dose was determined for each VOC, we evaluated the VOC response against polyclonal antibodies obtained from high titer HCP in a passive immunization study. The objective of the study was to assess the efficacy of antibodies derived from the ancestral strain on emerging VOC since binding and neutralizing antibodies against SARS-CoV-2 are the main correlates of protection for measuring immunity against SARS-CoV-2. Passive immunization of HCP and challenge using ancestral strain, Alpha, Beta or Delta resulted in protection against ancestral strain (100% survival), partial protection against Alpha (60%), and no protection against Beta or Delta challenge (0% survival). Survival outcomes of passive immunization and VOC challenge were also reflected on disease outcomes, viral RNA levels in the lung, brain, and nasal wash (Delta challenge only), and lung pathology. Despite poor outcomes, human RBD and nucleocapsid IgG levels remained stable in the serum and lung in the HCP treated and VOC challenged animals. Therefore, the VOC challenge mouse model established in this study was further used to study vaccine efficacy. Additionally, the HCP passive immunization study demonstrated to us that antibodies generated against the ancestral strain may not protect against VOC. Therefore, to better improve vaccine efficacy against VOC, Beta specific RBD antigens were utilized to study the efficacy of a VLP delivery approach in a murine challenge model. In this study, vaccines were formulated with RBD from either the ancestral strain (Wu) or Beta VOC conjugated to Hepatitis B surface antigen (HBsAg) VLP and adjuvanted with Aluminum hydroxide (Alum) or Squalene-in-water emulsion (SWE) and compared against Pfizer mRNA vaccine. Overall, all RBD-VLP vaccines generated RBD binding antibodies against multiple VOC RBD, broadly neutralizing antibodies against VOC RBD, decreased viral burden in the lung and brain, and lowered inflammation in the lung similar to Pfizer mRNA. However, only Beta and Wu RBD VLP adjuvanted with Alum, and Beta RBD VLP adjuvanted with SWE were able to protect mice (100% survival) against both Alpha and Beta challenge. Next, we evaluated intranasal (IN) vaccination as an approach to improve vaccine efficacy against SARS-CoV-2. We developed a prototype RBD vaccine conjugated to a diphtheria toxoid carrier protein Economical CRM197 (EcoCRM) and adjuvanted with a toll-like receptor agonist 4 (TRL4), Bacterial Enzymatic Combinatorial Chemistry (BECC) called BReC-CoV-2 (BECC+ RBD-EcoCRM COVID-19 vaccine). Overall, IN immunization with BReC-CoV-2 resulted in protection against SARS-CoV-2, decreased viral burden in the lung, brain and nasal wash, generated high levels of RBD IgG in the serum and lung that were capable of neutralizing VOC RBD, as well as induced mucosal IgA in the lung and nasal wash compared to intramuscular (IM) vaccination of BReC-CoV-2. Furthermore, heterologous IN prime and IM boost strategy with BReC-CoV-2 resulted in protection (100% survival) against a lethal Delta challenge. Altogether, the three approaches to improve vaccine efficacy demonstrated that the addition of VOC vaccine antigens accompanied with immunostimulatory adjuvants can improve vaccine responses to VOC and intranasal immunization can enhance vaccine protection by inducing mucosal antibody responses at the site of infection. Together, these vaccine approaches can help improve vaccine efficacy against emerging VOC in future COVID-19 vaccines

Targeted delivery of anti-cancer drugs by MS2 virus-like particles

Problems associated with poor pharmacokinetics and biodistribution, as well as toxic off-target effects, limit the curative potential of most anti-cancer drugs. This has prompted the development of nanoparticulate drug delivery systems to impart both more favourable pharmacological properties and precise tumour targeting. The vast number of formulations, ranging from fully synthetic delivery systems to ones derived from natural sources, currently undergoing clinical trials or preclinical testing underlines the significance of this field. This project is a proof-of-concept investigation into the feasibility and effectiveness of a novel drug delivery system, based on virus-like particles (VLPs) of the MS2 bacteriophage. Doxorubicin (Dox) and an anti-BCL2 siRNA were used as model drug cargos. They were packaged inside MS2 VLPs either by chemical infusion, or via covalent attachment to an MS2 packaging signal, TR, respectively. An average loading of ~10 molecules of siRNA or ~110 molecules of Dox per VLP was achieved. Packaged cargos remained stably encapsidated; the siRNA was protected from nuclease degradation. VLPs were surface decorated with polyethylene glycol (PEG), and tumour-targeting ligands, human transferrin (Tf) or A9L, an RNA aptamer that targets prostate-specific membrane antigen (PSMA). Extensive PEGylation was achieved (~97% of coat proteins), and each VLP displayed on average ~7 molecules of Tf or ~16 molecules of A9L. PEGylation significantly reduced the non-specific cellular uptake of VLPs, and antibody binding. Further addition of tumour-targeting ligands facilitated the specific delivery of drug cargos to targeted cancer cells in culture, likely via receptor-mediated endocytosis, and induced significant cytotoxicity with an LC50 of ~10 nM for siRNA and ~800 nM for Dox. Importantly, negligible toxic effects were observed in the presence of excess free targeting ligands, or with non-targeted control cell lines. Furthermore, the cellular uptake of VLPs did not appear to induce any off-target effects. MS2 VLPs continue to show promise as a robust, flexible and effective drug delivery system. This project highlights the versatility of VLPs for displaying a range of useful ligands on their surface, as well as packaging various therapeutic cargos, and demonstrated their ability to specifically deliver drugs to targeted cancer cells. Though further studies are required, the work presented here is an important step towards fully realising the potential of this drug delivery system

Food Additive

A food additive is defined as a substance not normally consumed as a food in itself and not normally used as a characteristic ingredient of food whether or not it has nutritive value. Food additives are natural or manufactured substances, which are added to food to restore colors lost during processing. They provide sweetness, prevent deterioration during storage and guard against food poisoning (preservatives). This book provides a review of traditional and non-traditional food preservation approaches and ingredients used as food additives. It also provides detailed knowledge for the evaluation of the agro-industrial wastes based on their great potential for the production of industrially relevant food additives. Furthermore the assessment of potential reproductive and developmental toxicity perspectives of some newly synthesized food additives on market has been covered. Finally, the identification of the areas relevant for future research has been pointed out indicating that there is more and more information needed to explore the possibility of the implementation of some other materials to be used as food additives