Investigating the host-directed therapeutic potential of curdlan-functionalised PLGA nanoparticles in the treatment of intracellular Mycobacterium tuberculosis

Du Plessis, Su-Mari (2020-03)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Despite the availability of antibacterial agents, tuberculosis (TB) remains the leading cause of death by a single infectious agent globally, accounting for millions of deaths annually. The high mortality and morbidity rates are associated with antibiotic resistance and tolerance. Further, Mycobacterium tuberculosis, the causative agent of TB, has successfully evolved various strategies to evade an antibacterial immune response. M. tuberculosis infection is a continuous interaction between the host and the bacterium. An improved fundamental understanding of how this bacterium can survive within host immune cells has paved the way for the development of host directed therapies (HDT) that utilize a different approach to conventional antibiotics; and such strategies are desperately required for the eradication of TB. HDT is an approach targeted at the host immune system instead of M. tuberculosis itself. By targeting the host immune system, there is potential to restrict the possible acquisition of genetic resistance, since the bacteria itself is not the target. To favour a protective immune response at the molecular level, immunotherapeutic nanoparticles (NPs) are being exploited. NPs are particulate structures at the nanoscale (1 – 1000 nm) with the ability to target specific cells, with such targeting achieved through modification of the surface of the NP with bioactive ligands. By targeting specific cells, the NPs can localize at the pathogen infected site and elicit an antibacterial response through ligand-receptor interactions. In this study, to target M. tuberculosis-infected macrophages (primary host immune cells), poly(lactide- co-glycolide) (PLGA) NPs were functionalised with curdlan, an immune-stimulatory polysaccharide known to target the dectin-1 receptor expressed on macrophages. Curdlan-dectin-1 interactions are known to stimulate the NF-kβ pathway that produces bactericidal factors such as oxidative species and pro-inflammatory cytokines and activate downstream pathways capable of killing intracellular bacilli. Thus, it is hypothesized that the introduction of curdlan-functionalised PLGA (C-PLGA) NPs to M. tuberculosis infected macrophages will stimulate the cells, leading to the death of the intracellular bacilli. Four different formulations were synthesized containing different curdlan loads and these included PLGA, 2%, 5% and 8% w/w C-PLGA NPs. Dynamic light scattering data confirmed the particles to be in the nano-size range with a negative zeta potential. The cytotoxicity of the NPs towards RAW264.7 macrophages was assessed using the MTT assay over 72h. Macrophages infected with M. tuberculosis ΔleuDΔpanD::pMV306hsp+lux expressing the bacterial luciferase gene, were used to assess the killing efficacy of the NPs (using luminescence as a proxy for cell numbers, with confirmatory colony forming unit (CFU) plating). While CFU data showed a trend towards reduction in M. tuberculosis bacterial numbers compared to untreated control following NP treatment, interpretation of results was cofounded by possible macrophage lysis, and this will require further investigation. Cytokine quantification highlighted the immune stimulating capabilities of the curdlan-functionalised NPs and upregulation of TNF-α was detected after 72h. The findings of this study support the hypothesis that C-PLGA NPs can stimulate M. tuberculosis infected macrophages, potentially leading to death of intracellular M. tuberculosis. This demonstrates the potential of C-PLGA NPs to be further developed as a HDT for TB.

AFRIKAANSE OPSOMMING: Ten spyte van beskikbare antibakteriële middels, bly tuberkulose (TB) die grootste oorsaak van sterftes deur 'n enkele infektiewe agent wêreldwyd en lei tot miljoene sterftes jaarliks. Die hoë mortaliteit en morbiditeit syfers word geassosieer met antibiotika weerstandigheid en verdraagsaamheid. Verder het Mycobacterium tuberculosis, die oorsaak van TB, verskeie strategieë ontwikkel om 'n antibakteriële immuunrespons suksesvol te ontduik. M. tuberculosis infeksies is 'n deurlopende interaksie tussen die gasheer en die bakterieë. ‘n Goeie begrip oor hoe hierdie bakterieë kan oorleef binne die gasheer se immuunselle het die weg gebaan vir die ontwikkeling van gasheer-gerigte terapie wat 'n ander benadering tot konvensionele antibiotika gebruik. Gasheer-gerigte terapie is 'n benadering wat direk gerig is op die gasheer se immuunsisteem in plaas van M. tuberculosis self. Deur die gasheer se immuunstelsel te teiken, is dit moontlik om die ontwikkeling van genetiese weerstand te beperk, aangesien die bakterieë self nie geteikin word nie. Om die immuunrespons op ‘n molekulêre vlak te kan beheer, word immunoterapeutiese nanopartikels (NPs) ingespan. NPs is baie klein strukture op die nanoskaal (1 – 1000 nm) met die vermoë om spesifieke selle te teiken. Dit word moontlik gemaak deur die oppervlak van die NP met bioaktiewe molekules te verweisig. Dus, deur spesifieke selle te teiken, kan die NPs ‘n gelokaliseerde antibakteriële reaksie ontlok deur middel van ligand-reseptor interaksies. Om M. tuberculosis-geïnfekteerde makrofage te teiken (primêre gasheer sel) word poly(lactide-co- glycolide) (PLGA) NPs geweisig met curdlan, 'n immuun-stimulerende polisakkaried gerig to die dectin- 1 reseptor op die oppervlak van makrofage. Curdlan-dectin-1 interaksies stimuleer die NF-kβ weg wat lei tot die vrystellling van verskeie antibakteriële faktore soos oksidatiewe spesies en pro- inflammatoriese molekules en gevolgelik verskeie paaie stroomaf aktiveer wat in staat is om intrasellulêre patogene dood te maak. Die hipotese is dus dat die aanwending van curdlan-geweisigde PLGA (C-PLGA) NPs geïnfekteerde immunselle sal stimuleer, wat lei tot die dood van die intrasellulêre bakterieë. Vier verskillende NP formulerings is geproduseer met verskillende hoeveelhede curdlan en sluit in PLGA, 2%, 5% en 8% C-PLGA NPs. Dinamiese lig verstrooiing (DLV) data het bevestig dat die NPs van nano-grootte is met 'n negatiewe zeta potensiaal en stabiel voorkom in ‘n oplossing. Die sellulêre toksisiteit van die NPs is geassesseer oor 72 uur op RAW264.7 makrofage deur middel van die MTT toets. Makrofage is geïnfekteer met M. tuberculosis ΔleuDΔpanD::pMV306hsp + Lux wat ons in staat stel om die groei inhiberende potensiaal van die NPs te meet deur verandering in luminessensie te gebruik as ‘n aanduiding van seldood gevolg deur die tel van kolonie vormende eenhede (KVE). Terwyl KVE data 'n afname in M. tuberculosis selgetalle toon vir die NP behandelde selle in vergelyking met luminessensie is die resultate verwarrend moontlik as gevolg van die makrofage wat liseer en dus sal dit verdere ondersoek vereis. Sitokien kwantifisering het die immuunstimulerende vermoëns van die curdlan-verweisigde NPs uitgelig en ‘n verhoogde uitdrukking van TNF-α is na 72 uur opgespoor. Die bevindinge van hierdie studie ondersteun die hipotese dat C-PLGA NPs ‘n immuunresponse kan stimuleer in M. tuberculosis geïnfekteerde makrofage en dus lei tot 'n dood van intrasellulêre patogene. Dit toon die potensiaal van C-PLGA-NPs om verder as 'n gasheer-gerigte terapie vir TB ontwikkel te word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108481
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