Polymer-coated magnetic nanoparticles and modified polymer nanofibers for the efficient capture of Mycobacterium tuberculosis (Mtb)

Smit, Marica (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: The World Health Organization (WHO) determined that 10.4 million people died of tuberculosis (TB) in 2015 which makes TB the number one cause of death from a preventable infectious disease worldwide. Mycobacterium tuberculosis (Mtb) is the causative pathogen of TB and a frequent lack of clinical symptoms hampers the pathogen’s detection. Current diagnostic tests are limited when applied to low populations of bacteria in biological fluids, such as blood. A large volume of biological fluid is needed for a positive diagnosis. Obtaining multiple samples are, however, difficult, especially from children under six years. A smaller amount of biological fluid will be needed if the Mtb can be captured and concentrated within the sample. Polymer coated superparamagnetic magnetite nanoparticles (SPMNs) with affinity for the pathogen can be used as capturing substrates for Mtb followed by diagnosis via existing microscopy methods such as fluorescence microscopy (FM). In this thesis, modified chitosan and modified poly(styrene-alt-maleic anhydride) (SMA) were synthesized and utilized to coat SPMNs as well as electrospun into nanofibers to form potential Mtb capturing substrates. Chitosan and SMA were modified to from quaternary derivatives which can possibly interact with the Mtb cell wall. The nano-substrates were also surface functionalized with a carbohydrate binding protein, namely Concanavalin A (Con A), which can bind to the Mtb cell wall. Chitosan coated SPMNs were synthesized by in situ co-precipitating Fe2+ and Fe3+ with chitosan followed by further modification. SMA coated SPMNs were synthesized by activating the iron oxide core with 3-aminopropyl(triethoxysilane) (3-APTES) followed by further modification. Polymer nanofibers were electrospun via single needle electrospinning. The chitosan derivatives were electrospun into nanofibers by blending with non-ionogenic polymers, viz. polyvinyl alcohol (PVA), polylactide (PLA), polycaprolactam (Nylon 6), polyethylene oxide (PEO) and polyvinyl pyrrolidone (PVP) to facilitate electrospinning. The nano-substrates were evaluated for their affinity and thus capturing capabilities utilizing the mCherry fluorophore tagged bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis, a live attenuated Mtb-mimic. A preliminary nanofiber affinity study was conducted to determine which polymer-and-functional-moiety combination had the highest affinity for the bacteria utilizing FM (fluorescence microscopy). Quaternary SMA (SMI-qC12) had the highest affinity for BCG-mCherry (through electrostatic and hydrophobic interactions) followed by Con A immobilized chitosan (CS-EDC-Con A). The SPMNs were coated with three different polymer loadings and a dilution study performed to determine the limit of detection. The 0.9 g loaded SMI-qC12 SPMNs had the highest affinity for BCG-mCherry determined via FM and TEM (transmission electron microscopy).

AFRIKAANSE OPSOMMING: Die wêreldgesondheidsorganisasie (WGO) het bepaal dat 10.4 miljoen mense gesterf het a.g.v tuberkolose (TB) in 2015 wat TB dus die vernaamste oorsaak van dood aan voorkombare aansteeklike siektes maak wêreldwyd. Mycobacterium tuberculosis (Mtb) is die patogeen wat TB veroorsaak en ‘n gereelde gebrek aan kliniese simptome belemmer die opsporing van die patogeen. Huidige diagnostiese toetse is beperk wanneer toegepas word op lae populasies van bakterieë in biologiese vloestowwe, soos bloed. ‘n Groot volume biologiese vloeistof word benodig vir ‘n positiewe diagnose. Die verkryging van veelvuldige monsters is egter moeilik, veral van kinders onder ses jaar. ‘n Kleiner hoeveelheid biologiese vloeistof word benodig as die Mtb vasgevang en gekonsentreer kan word binne die monster. Polimeer bedekte superparamagnetiese magnetiet nanopartikels (SPMNs) met ‘n affiniteit vir die patogeen kan gebruik word as vasvangingsubstrate vir Mtb gevolg deur diagnose via bestaande mikroskopie metodes soos fluoresensie mikroskopie (FM). In hierdie tesis is gemodifiseerde chitosan en gemodifiseerde poli (styreen-alt-maleïensanhidried) (SMA) gesintetiseer en gebruik om SPMNs te bedek asook om nanovesels te elektrospin om potensiële Mtb vasvangingsubstrate te vorm. Chitosan en SMA is gemodifiseer om kwaternêre afgeleides te vorm wat moontlik ‘n interaksie kan hê met die Mtb selwand. Die nano-substrate is ook oppervlak gefunktionaliseer met ‘n koolhidraat bindende proteïen, naamlik Concanavalin A (Con A), wat kan bind aan die Mtb selwand. Chitosan bedekte SPMNs was gesintetiseer deur in situ mede-neerslag van Fe2+ en Fe3+ met chitosan gevolg deur verdere modifikasie. SMA bedekte SPMNs is gesintetiseer deur aktivering van die ysteroksied kern met 3-aminopropiel(trietoksiesilaan) (3-APTES) gevolg deur verdere modifikasie. Polimeer nanovesels was geëlektrospin via die enkelnaald elektrospin tegniek. Die chitosan afgeleides is tot nanovesels geëlektrospin deur vermenging met nie-ionogeniese polimere, nl. poliviniel alkohol (PVA), polilaktied (PLA), polikaprolaktaam (Nylon 6), poliëtileen oksied (PEO) and poliviniel pirrolidoon (PVP) om elektrospin te fasiliteer. Die nano-substrate is geëvalueer vir hul affinitiet en dus vasvangingsvermoë d.m.v die mCherry fluorofoor gemerkte bacillus Calmette-Guérin (BCG) stam van Mycobacterium bovis, ‘n lewendige verswakte Mtb mimiek. ‘n Voorlopige nanovesel affiniteitstudie is uitgevoer om te bepaal watter polimeer-en-funksionele-group kombinasie die hoogste affinitiet het vir die bakterieë d.m.v FM (fluoresensie mikroskopie). Kwaternêre SMA (SMI-qC12) het die hoogste affiniteit gehad vir BCG-mCherry (deur elektrostatiese en hidrofobiese interaksies) gevolg deur Con A geïmmobiliseerde chitosan (CS-EDC-Con A). Die SPMNs is bedek met drie verskillende polimeer ladings en ‘n verdunningstudie is uitgevoer om die opsporingsperk te bepaal. Die 0.9 g bedekte SMI-qC12 het die hoogste affiniteit gehad vir BCG-mCherry soos bepaal via FM en TEM (transmissie elektron mikroskopie).

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