Engineering carbon nanotubes as therapeutic nanocarriers of Tulbaghia violacea, Annona muricata, Dicoma capensis and Dodonaea viscosa plant-based extracts, targeting breast and colorectal cancer

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Stellenbosch : Stellenbosch University
ENGLISH ABSTRACT: Cancer is one of the most prevalent diseases globally and it is characterized by uncontrolled rapid cell division and differentiation. Lack of tumour specificity, dose-related toxicity and low bioavailability of chemotherapy drugs are major hindrances to cancer treatment. Nanotechnology has given the platform to selectively interact with cancerous cells and increase cellular uptake and drug localization. Functionalisation of nanoparticles can be done to recognize cancer cells and giving accurate and selective drug delivery which does not interact with healthy cells. In order to develop more efficient therapeutic regimes, a better understanding of the type of nanoparticles suitable for drug delivery is required. Over the past years, carbon nanotubes have been used as nanocarriers to transport anticancer drugs, genes, and proteins for chemotherapy. Furthermore, the possibility of conjugating carbon nanotubes with anticancer plant-based drugs creates advanced therapeutic applications. Therefore, the aim of this project was to develop a single-walled carbon nanotube (SWCNT) nanocarrier bio-conjugated with plant-based bioactive compounds which can target can- cer cells specifically. The first phase of the study involved purification and functionalisation of carbon nanotubes. Hydrochloric acid was used to purify the carbon nanotubes and functionalisation was done with polyethylene glycol (PEG) and folic acid (FA). Fourier transform infrared (FTIR) spectroscopy was used to confirm functionalisation. Four plants were identified and used, namely Annona muricata, Dodonaea viscosa, Dicoma capensis and Tulbaghia violacea. Two plant extraction methods were assessed, and the closed loop extraction method obtained the most bioactive compounds from the plant extracts. The results were confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis. This was followed by bio-conjugation of the functionalised carbon nanotubes with bioactive compounds from the four plants. Ultraviolet–visible (UV-Vis) spectroscopy was used to confirm bio-conjugation. In vitro cytotoxicity studies were undertaken to assess the effects of bioactive compounds and bio-conjugates in breast cancer cell lines (MCF-7 and MDA-MB-231), a colon cancer cell line (HT-29) and a non-tumorigenic breast epithelial cell line (MCF-12A). The in vitro cytotoxicity results showed a low cell viability for cancer cell lines whilst that for normal cells remained higher. The half-maximal inhibitory concentration (IC50) values were determined, and all plants showed a value lower than 30 μg/ml. The selectivity index (SI) of the plant extracts was also calculated and all plants showed a high SI value greater than 2. The bio-conjugates showed a higher cell viability in normal cells and a lower cell viability in cancer cells compared to plant extracts alone, which was due to the conjugation with carbon nanotubes improving selectivity and efficacy. Furthermore, the bio-conjugate with all four plant extracts mixed had the lowest cell viability in all cancer cell lines indicating synergism. The mechanisms of cellular uptake were determined by fluorescence microscopy and it was observed that folate receptor mediated endocytosis and caveolae mediated endocytosis both took place for bio-conjugate cellular internalization. The type of cell death occurring in cancer cells was determined by flow cytometry studies. It was confirmed that both apoptosis and necrosis took place in the cancer cells. In conclusion, the carbon nanotubes were successfully used as nanocarriers and their bio-conjugation with plant based bioactive compounds improved efficacy and selectivity towards cancer cells in this study.
AFRIKAANSE OPSOMMING: Kanker is een van die mees algemene siektes wˆereldwyd en word gekenmerk deur onbeheerde vinnige seldeling en differensiasie. Gebrek aan tumorspesifisiteit, dosisverwante toksisiteit en lae biobeskikbaarheid van chemoterapiemiddels is groot hindernisse vir kankerbehandeling. Nanotegnologie het die platform verskaf om selektief met kankerselle om te gaan en sellulˆere opname en geneesmiddellokalisering te verhoog. Funksionalisering van nanopartikels kan gedoen word om kankerselle te herken en om akkurate en selektiewe geneesmiddellewering te gee wat nie met gesonde selle in wisselwerking tree nie. Om meer doeltreffende terapeutiese regimes te ontwikkel, is ’n beter begrip van die tipe nanopartikels wat geskik is vir geneesmiddelaflewering nodig. Oor die afgelope jare is koolstofnanobuise as nanodraers gebruik om kankermedisyne, gene en prote¨ıene vir chemoterapie te vervoer. Verder skep die moontlikheid om koolstofnanobuise met antikanker-plantgebaseerde middels te kombineer, gevorderde terapeutiese toepassings. Die doel van hierdie projek was dus om ’n enkelwand koolstofnanobuis (SWCNT) nanodraer te ontwikkel wat bio-gekonjugeer is met plantgebaseerde bioaktiewe verbindings wat kankerselle spesifiek kan teiken. Die eerste fase van die studie het die suiwering en funksionalisering van koolstofnanobuise behels. Soutsuur is gebruik om die koolstofnanobuise te suiwer en funksionalisering is met poli¨etileenglikol (PEG) en foliensuur (FA) gedoen. Fourier transform infrarooi (FTIR) spektroskopie is gebruik om funksionalisering te bevestig. Vier plante is ge¨ıdentifiseer en gebruik, naamlik Annona muricata, Dodonaea viscosa, Dicoma capensis en Tulbaghia violacea. Twee plantekstraksiemetodes is geassesseer, en die geslotelus ekstraksiemetode het die mees bio-aktiewe verbindings uit die plantekstrakte verkry. Die resultate is bevestig deur vloeistofchromatografie-massaspektrometrie (LC-MS) analise. Dit is gevolg deur biokonjugasie van die gefunksionaliseerde koolstofnanobuise met bio-aktiewe verbindings van die vier plante. Ultraviolet-sigbare (UV-Vis) spektroskopie is gebruik om bio-konjugasie te bevestig. In vitro sitotoksisiteitstudies is gedoen om die effek van bio-aktiewe verbindings en bioconjugate in borskankersellyne (MCF-7 en MDA-MB-231), ’n kolonkankersellyn (HT-29) en ’n nie-tumogeniese borsepiteelsellyn (MCF-12A) te bepaal. Die in vitro sitotoksisiteit resultate het ’n lae sellewensvatbaarheid vir kankersellyne getoon, terwyl dit vir normale selle ho¨er gebly het. Die half-maksimale inhiberende konsentrasie (IC50) waardes is bepaal, en al die plante het ’n waarde laer as 30 μg/ml getoon. Die selektiwiteitsindeks (SI) van die plantekstrakte is ook bereken en alle plante het ’n ho¨e SI-waarde groter as 2 getoon. Die bio-konjugate het ’n ho¨er sellewensvatbaarheid in normale selle en ’n laer sellewensvatbaarheid in kankerselle getoon, in vergelyking met plantekstrakte alleen, wat te wyte was aan die kombinasie met koolstofnanobuise wat selektiwiteit en doeltreffendheid verbeter het. Voorts het die bio-konjugaat met al vier plantekstrakte gemeng die laagste sellewensvatbaarheid in alle kankersellyne gehad wat sinergisme aandui. Die meganismes van sellulˆere opname is deur fluoressensiemikroskopie bepaal en daar is waargeneem dat folaatreseptor-gemedieerde endositose en caveolae-gemedieerde endositose beide plaasgevind het vir bio-gekonjugeerde sellulˆere internalisering. Die tipe seldood wat in kankerselle voorkom, is deur vloeisitometrie-studies bepaal. Dit is bevestig dat beide apoptose en nekrose in die kankerselle plaasgevind het. Ten slotte, die koolstofnanobuise is suksesvol as nanodraers gebruik en hul bio-konjugasie met plantgebaseerde bio-aktiewe verbindings het doeltreffendheid en selektiwiteit teenoor kankerselle in hierdie studie verbeter.
Thesis (PhD)--Stellenbosch University, 2023.