Surface modification of styrene maleic anhydride nanofibers for efficient capture of Mycobacterium tuberculosis

Cronje, Lizl (2012-12)

Thesis (PhD)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: Tuberculosis (TB) is a major cause of morbidity and mortality across the world, affecting adults and children. Children infected with TB differ from adults, as their immunological and patho-physiological response to the disease is different. Although there are a variety of tests available for TB diagnosis, they have limitations when used to diagnose paediatric TB. Children are also unable to generate sputum spontaneously when required for the use in culture or microscopy as diagnostic method. Children however do produce sputum, containing the TB bacilli, which they swallow. If the TB bacilli can therefore be retrieved from the stomach and tested, TB can be diagnosed using gastric samples. In this thesis, a variety of styrene maleimide copolymer (SMI) derivatives were prepared as potential M. tuberculosis-capturing platforms. This was done by modifying poly(styrene-co-maleic anhydride) (SMA) with a variety of primary amine compounds, selected based on possible chemical interactions with the M. tuberculosis cell wall. All the prepared copolymer derivatives were electrospun into nanofibrous mats using the single needle electrospinning technique to yield SMI nanofibers, functionalized with different compounds. Some of the functionalized SMI nanofibers were prepared by surface-functionalization of the polymer nanofibers after electrospinning and some by modification of the polymer before electrospinning. Affinity studies were conducted at neutral and low pH between the different functionalized SMI nanofibers and two mycobacterium strains, namely the bacillus Calmette-Guérin strain of Mycobacterium bovis (BCG) and M. tuberculosis, to evaluate the surfaces of the modified SMI nanofibers as mycobacterium-capturing platforms. The successful capture of BCG onto the surfaces of the various functionalized nanofibers was confirmed by SEM and fluorescence microscopy (FM). Analysis of the SEM and FM images indicated that the SMI nanofibers, functionalized with a C12 aliphatic quaternary ammonium moiety (SMI-qC12), captured BCG the most effectively through a combination of ionic and hydrophobic interaction. Concentration and time studies revealed that the extent of this interaction was dependent on incubation time and concentration of BCG. The affinity studies with BCG also concluded that the polymer used for the nanofibrous-capturing platform should not be too hydrophobic in character as this caused poor wetting of the functionalized nanofibers, thus preventing close contact with the mycobacteria and a reduction in the capture effectivity of the polymer nanofibers. The successful capture of M. tuberculosis onto the SMI-qC12 nanofibrous surface was confirmed by FM, light microscopy (LM) and polymerase chain reaction (PCR). The extent of this interaction was dependent on the concentration of M. tuberculosis. The detection of M. tuberculosis using FM and LM as detection methods was simplified by the tendency of M. tuberculosis to clump together in clusters on the hydrophobic surface of the SMI-qC12 nanofibers. As a result of this clustering, FM and LM were therefore regarded as feasible detection methods to image M. tuberculosis on the surface of the SMI-qC12 nanofibers, even at relatively low concentration of M. tuberculosis.

AFRIKAANSE OPSOMMING: Tuberkulose (TB) is 'n groot oorsaak van morbiditeit en mortaliteit regoor die wêreld en affekteer volwassenes en kinders. Kinders wat met TB geïnfekteer is, se immunologiese en patofisiologiese reaksie op die siekte verskil van die van volwassenes en dit het belangrike implikasies vir die diagnose van TB in kinders. Alhoewel daar 'n verskeidenheid van toetse beskikbaar is vir die diagnose van TB, het hulle beperkings wanneer dit gebruik word om pediatriese TB te diagnoseer. Kinders kan ook nie spontaan sputum produseer as dit nodig is vir die gebruik in kultuur of mikroskopie as diagnostiese metode. Kinders produseer egter wel sputum, wat die TB basille bevat, wat hulle dan insluk. As die TB basille uit die maag versamel kan word en getoets kan word, kan TB gediagnoseer word met behulp van maag monsters. In hierdie tesis is 'n verskeidenheid van stireen maleimied kopolimeer (SMI) afgeleides voorberei as potensiële Mycobacterium tuberkulose (Mtb)-vaslegging platforms. Dit is gedoen deur die modifikasie van poli(stireen-ko-maleïen anhidried) (SMA) met 'n verskeidenheid primêre amien verbindings as oppervlak-funksionaliseringsagente. Hierdie primêre amien verbindings is gekies op grond van moontlike chemiese interaksies met die Mtb selwand. Al die voorbereide kopolimeer afgeleides is elektrogespin in nanoveselagtige matte met behulp van die enkel-naald elektrospin tegniek om SMI nanovesels te lewer wat gefunksionaliseer is met verskillende verbindings. Sommige van die gefunksionaliseerde SMI nanovesels is berei deur oppervlak-funksionalisering van die polimeer nanovesels na elektrospin, en sommige deur die modifikasie van die polimeer voor elektrospin. Affiniteitstudies is uitgevoer, by neutrale en lae pH, tussen die verskillende gefunksionaliseerde SMI nanovesels en twee mikobakterium rasse, naamlik die basillus Calmette-Guérin ras van Mycobacterium bovis (BCG) en M. tuberculosis, om die oppervlaktes van die gewysigde SMI nanovesels te evalueer as mikobakterium-vaslegging platforms. Ontleding van die SEM en FM beelde het aangedui dat die SMI nanovesels, gefunksionaliseer met 'n C12 alifatiese kwaternêre ammonium groep (SMI-qC12), BCG die doeltreffendste vasgevang het deur 'n kombinasie van ioniese en hidrofobiese interaksie. Konsentrasie- en tydstudies tussen BCG en SMI-qC12 het aangedui dat die omvang van hierdie interaksie afhanklik is van inkubasietyd en konsentrasie van BCG. Die affiniteitstudies met BCG het ook aangedui dat die polimeer wat gebruik word vir die nanoveselagtige-vaslegging platform nie te hidrofobiese moet wees nie, aangesien dit swak benatting van die gefunksionaliseerde nanovesels veroorsaak, en dus noue kontak met die mikobakterieë voorkom met ʼn gevolglike vermindering in die vasvang-effektiwiteit van die polimeer nanovesels. Die suksesvolle vasvang van M. tuberculosis op die SMI-qC12 nanovesels is bevestig deur FM, lig mikroskopie (LM) en polimerase kettingreaksie (PKR). Die opsporing van Mtb deur die gebruik van FM en LM as opsporingmetodes is vergemaklik deur die tendens van Mtb om in groepies saam te pak op die hidrofobiese oppervlak van die SMI-qC12 nanovesels. As gevolg van hierdie groepering, is FM en LM dus haalbare opsporingmetodes om M. tuberculosis op die oppervlak van die SMI-qC12 nanovesels waar te neem, selfs by relatief lae konsentrasie van M. tuberculosis.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/71923
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