Functionalized polymer nanofibrous substrates as capturing platforms for mycobacteria

du Plessis, Anja Nicola (2016-03)

Thesis (MSc)--Stellenbosch University, 2016

Thesis

ENGLISH ABSTRACT : Tuberculosis (TB) remains one of the world’s deadliest communicable diseases. In 2013, an estimated 9.0 million people developed TB and 1.5 million died from the disease. The treatment of children with TB is often not considered a priority by tuberculosis control programmes. Paediatric TB is frequently more difficult to diagnose due to non-specific clinical and radiological features as opposed to adult TB cases that are often easily recognizable and accompanied by a positive sputum smear test. Most cases of childhood TB are paucibacillary, and therefore mycobacterial culture of specimens is required to optimise diagnostic yield, which can take weeks. If the TB bacilli can be concentrated in specimens, this problem could be circumvented and rapid diagnosis could be possible using existing microscopy methods. In this thesis, styrene maleimide copolymer (SMI) derivatives were synthesized and electrospun to develop nanofibrous substrates with an affinity for the mycobacterial cell wall of Mycobacterium tuberculosis (Mtb) to potentially act as capturing platforms for these bacilli. SMA was modified with a variety of low molecular weight modification agents that were chosen based on their known or possible affinity for the mycobacterial cell wall before being electrospun into nanofibrous membranes. Prefabricated SMA nanofibers were also surface-functionalized with a lectin-binding protein, namely Concanavalin A (Con A). Affinity studies were carried out between the modified polymer nanofibers and an attenuated strain of Mtb, namely Mycobacterium bovis bacillus Calmette-Guérin (BCG) to evaluate the ability of each polymer nanofibrous substrate to capture BCG at decreasing concentrations. The successful capture of BCG onto the substrates was confirmed using fluorescence microscopy (FM). Analysis of the FM images revealed that SMA functionalized with Con A captured BCG the most effectively due to the saccharide binding properties of the protein. This interaction is specific between BCG and the Con A protein due to the mannose binding ability of Con A and the mannose molecules present on the outer cell surface of BCG. SMA modified with aliphatic quaternary ammonium moieties of chain lengths C8-C12 also showed exceptional capturing abilities through a combination of ionic and hydrophobic interactions. This interaction is non-specific as it is only dependent on the electrostatic and hydrophobic-hydrophobic interaction between BCG and the polymer nanofibrous surfaces. These interactions were visible even at the lowest tested BCG concentration.

AFRIKAANSE OPSOMMING : Tuberkulose (TB) bly een van die wêreld se dodelikste oordraagbare siektes. In 2013 het ‘n geskatte 9.0 miljoen mense TB ontwikkel en 1.5 miljoen het gesterf as gevolg van die siekte. Die behandeling van kinders met TB word dikwels nie as ‘n prioriteit beskou deur tuberkulose programme nie. Pediatriese TB is dikwels moeilik om te diagnoseer as gevolg van nie-spesifieke kliniese en radiologiese kenmerke in teenstelling met volwasse TB-gevalle wat dikwels maklik herkenbaar is en vergesel word deur ‘n positiewe sputum smeer toets. Die meeste gevalle van kinder TB word geken aan die teenwoordigheid van min basille, daarom word mikobakteriële kultuur van monsters benodig om diagnostiese opbrengs te optimaliseer. Hierdie prosedure kan weke neem. Indien die TB-basille gekonsentreer kan word vanuit die monsters kan hierdie probleem omseil word en sodoende ‘n vinnige diagnose bewerkstellig deur gebruik te maak van bestaande mikroskopiese metodes. In hierdie tesis is stireen maleimied kopolimeer (SMI) afgeleides gesintetiseer en elektrogespin om nanoveselagtige substrate te ontwikkel met ‘n affiniteit vir die mikobakteriële selwand van Mycobacterium tuberkulose (Mtb) wat potensieël as vaslegging platforms vir hierdie basille kan optree. SMA is gemodifieer met ‘n verskeidenheid van lae molekulêre massa verbindings wat gekies is op grond van hul bekende of moontlike affiniteit vir die mikobakteriële selwand, waarna die polimere elektrogespin is in nanoveselagtige membrane. Voorafvervaardigde SMA nanovesels was ook gemodifiseer met ‘n koolhidraat-bindende proteïen, naamlik Concanavalin A (Con A) deur oppervlak-funksionalisering. Affiniteitstudies is uitgevoer tussen die gemodifiseerde polimeer nanovesels en ‘n verswakte stam van Mtb, naamlik Mycobacterium bovis bacillus Calmette-Guérin (BCG) om die vermoë van elke polimeriese nanoveselagtige substraat om BCG vas te vang, te evalueer met vermindering van BCG konsentrasie. Die suksesvolle vasvang van BCG op die substrate is bevestig met behulp van fluoressensie mikroskopie (FM). Ontledig van die FM beelde het aangedui dat SMA gemodifiseer met Con A as die doeltreffendste substraat opgetree het deurdat hierdie vesels die meeste BCG vasgevang het as gevolg van die sakkaried-bindende eienskappe van die proteïen. Hierdie interaksie is spesifiek tussen BCG en die Con A proteïen as gevolg van die mannose-bindingsvermoë van Con A en die mannose molekules teenwoordig op die buitenste seloppervlak van BCG. SMA wat gemodifiseer is met alifatiese kwaternêre ammonium groepe met kettinglengtes van C8-C12 het ook uitsonderlike vasvang vermoëns getoon deur middel van ‘n kombinasie van ioniese en hidrofobiese interaksies. Hierdie interaksie is nie-spesifiek omdat dit slegs afhanklik is van die elektrostatiese en hidrofobiese-hidrofobiese interaksie tussen BCG en die polimeriese nanoveselagtige oppervlaktes. Hierdie interaksies was selfs by die laagste konsentrasie BCG sigbaar.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98452
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