Modified chitosan nano-substrates for mycobacterial capture

Fortuin, Lisa (2015-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Tuberculosis (TB) is one of the world’s deadliest diseases, with one third of the population being infected by it. The diagnosis of active tuberculosis entails finding and identifying Mycobacterium tuberculosis (Mtb), the causative pathogen in a specimen of bodily fluid from the patient. Multiple samples will improve the diagnostic yield and specimen volumes should therefore be as large as possible, which is often challenging for patients and especially younger children. Alternatively, a smaller volume could be required if there was a manner in which to concentrate the bacteria within a specimen, through use of a substrate which has an affinity for the pathogenic species. Polymers having intrinsic cellular activity are of interest as such substrates, one such being the natural polysaccharide, chitosan. In this thesis, a variety of modified chitosan derivatives were prepared as potential Mtb-capturing substrates. This was achieved by modifying chitosan with a variety of moieties, selected based on possible interactions with the Mtb cell wall, to render various quaternary ammonium salts of the polymer chitosan. The quaternized chitosan derivatives were then used to synthesize nano-substrates having an affinity for Mtb. Polymer coated superparamagnetic magnetite nanoparticles (SPMNs) were synthesized via an in situ co-precipitation technique, in which modified chitosan is able to chelate with the metal core. Polymer nanofibers were also electrospun via the electrospinning technique. The prepared derivative, N-trimethylammonium chitosan chloride (TMC), was electrospun into nanofibers by blending with suitable non-ionogenic polymers, namely polyvinyl alcohol (PVA), polyethylene oxide (PEO), polyvinyl pyrrolidone (PVP) and polyacrylamide (PAM), required to facilitate nanofiber formation. Affinity studies were conducted between the modified chitosan nano-substrates and the bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis, the attenuated Mtb-mimic bacteria, for evaluation as mycobacterium capturing substrates. The successful capture of BCG onto the surfaces of the various modified chitosan nanofibers and modified chitosan coated superparamagnetic nanoparticles was confirmed by fluorescence microscopy (FM), light microscopy (LM), transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM). Analysis of the FM, TEM and FE-SEM images indicated that the chitosan coated nanoparticles functionalized with a C12 aliphatic quaternary ammonium moiety (CS-qC12), captured the most BCG through a combination of ionic and hydrophobic interaction. TMC blended with PVA, to produce nanofibers crosslinked with genipin, were found to have the strongest interaction with BCG of the nanofibrous mats tested. These findings were corroborated by water contact angle measurements, which established that PVA was the least hydrophilic of the non-ionogenic polymers and had hydrogen bond donating groups only, factors influencing the cellular adhesive properties of affinity substrates.

AFRIKAANSE OPSOMMING: Tuberkulose (TB) is een van die wêreld se mees dodelikste siektes, met ‘n derde van die bevolking wat geïnfekteer is daarmee. Ten einde aktiewe TB te diagnoseer moet Mycobacterium tuberculosis (Mtb), die voorsakende patogeen in ʼn monster van die pasiënt se liggaamlike vloeistof, gevind en ïdentifiseer word. Veelvuldige monsters sal die diagnotiese opbrengs verhoog en monster volumes moet dus so groot as moontlik wees wat dikwels ʼn uitdaging vir pasiënte en veral jonger kinders kan bied. Alternatiewelik kan ʼn kleiner monster van die pasiënt vereis word indien daar ʼn manier was om die bakterieë in ʼn monster te konsentreer deur die gebruik van ʼn substraat wat ʼn affiniteit toon vir die patogeniese spesie. Polimere met ʼn intrinsieke sellulêre aktiwiteit, wek belangstelling as sodanige substraat, een synde die natuurlike polisakkaried, chitosan. In hierdie tesis is ʼn verskeidenheid gemodifiseerde chitosan afgeleides voorberei as potensiële Mtb-vaslegging substrate. Dit is gedoen deur chitosan te modifiseer met ʼn verskeidenheid funksionele groepe, gekies op grond van moontlike interaksies met die Mtb selwand, ten einde ʼn verskeidenheid kwaternêre ammonium soute van die chitosan polimeer te bekom. Die kwaternêre chitosan afgeleides is gevolglik gebruik om nano-substrate te sintetiseer wat ʼn affiniteit toon vir Mtb. Polimeer bedekte superparamagnetiese magnetiet nanopartikels (SPMNs) is gesintetiseer via ʼn in situ mede-neerslag metode, waarvolgens die gemodifiseerde chitosan polimere in staat is om met die metaal kern te chelaat. Polimeer nanovesels is ook geëlektrospin deur die elektrospin tegniek te gebruik. Die voorbereide afgeleide N-trimetielammonium chitosan chloried (TMC) is tot nanovesels geëlektrospin deur vermenging met geskikte nie-ionogeniese polimere, naamlik poliviniel-alkohol (PVA), polietilene-oksied (PEO), poliviniel-pirrolidoon (PVP) en poliakrielamied (PAM), wat vereis word ten einde nanovesels te produseer. Affiniteit studies is uitgevoer tussen die gemodifiseerde chitosan nano-substrate en die bacillus Calmette-Guérin (BCG) stam van Mycobacterium bovis, die verswakte Mtb-mimiek bakterieë vir evaluering as mycobakterium-vaslegging substrate. Die suksesvolle vasvang van BCG op die oppervlaktes van die verskillende gemodifiseerde chitosan nanovesels en gemodifiseerde chitosan bedekte SPMNs is bevestig deur fluoressensie mikroskopie (FM), lig mikroskopie (LM), transmissie elektron mikroskopie (TEM) en veld-emissie-skandering elektron mikroskopie (FE-SEM). Analise van die FM, TEM en FE-SEM beelde het getoon dat die chitosan bedekte nanopartikels met byvoeging van ʼn C12 alifatiese kwaternêre ammonium groep, die meeste BCG vasgevang het deur ʼn kombinasie van ioniese en hidrofobiese interaksie. TMC vermeng met PVA om nanovesels te vorm, gekruisbind met genipin, is gevind om die sterkste interaksie met BCG te toon. Hierdie bevindings is bevestig deur water-kontak-hoek-metings, wat getoon het dat PVA die minste hidrofilies van die nie-ionogeniese polimere was en slegs waterstof-binding skenkings groepe het, alles faktore wat die sellulêre bindingskwaliteite van affiniteit-substrate sal beïnvloed.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/97882
This item appears in the following collections: