The influence of biomimetic nanoparticles on macrophage phenotype and their anti-mycobacterial efficacy on macrophages.

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dc.contributor.advisorSampson, Samantha Leighen_ZA
dc.contributor.advisorDu Plessis, Nelitaen_ZA
dc.contributor.authorMaaruf, Shamsuddeen Yusufen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.en_ZA
dc.date.accessioned2024-02-15T08:18:27Zen_ZA
dc.date.accessioned2024-04-27T00:16:43Zen_ZA
dc.date.available2024-02-15T08:18:27Zen_ZA
dc.date.available2024-04-27T00:16:43Zen_ZA
dc.date.issued2024-02en_ZA
dc.descriptionThesis (MSc)--Stellenbosch University, 2024.en_ZA
dc.description.abstractENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) remains a major public health concern. Despite the availability of various treatment regimens, treatment failure is common, partly attributed to the phenomenon of bacterial persisters. In addition, repeated or lengthy treatments can potentially induce drugresistant strains of M. tuberculosis which further compound TB control. New approaches to TB therapy are therefore needed. To promote the intracellular killing of M. tuberculosis through pro-inflammatory responses (M1), a promising approach is the application of nanoparticles (NPs), which include lipid-polymer hybrid (LPN), to deliver drugs or immunomodulatory compounds such as Curdlan (CN) to macrophages. This represents a novel, host-based therapeutic approach for TB. While M1 polarized macrophages are thought to be the most effective in controlling M. tuberculosis infection, it has been shown that intracellular bacilli induce a shift to the M2 phenotype. There is no evidence that the introduction of NPs such as LPN conjugated with CN can induce a shift in macrophage phenotype or that the macrophage polarization state affects the antibacterial activity of NPs. In this study, we first set out to evaluate the anti-mycobacterial efficiency of CN-functionalized NPs in vitro, which had previously shown to be promising in the inhibition of mycobacterial growth intracellularly and in vivo in a parent study. Primary cells derived from the C57BL/6 mice bone marrow called bone marrow derived macrophages (BMDMs) were used for this study. We used an attenuated M. tuberculosis H37Rv laboratory strain with a reporter plasmid (pMV306hsp+Lux) to allow real-time monitoring of luminescence as a proxy for the bacterial load. Results from the real-time luminescence monitoring confirmed that the CNfunctionalized NPs is a good anti-mycobacteria agent as previously demonstrated in the parent study. We further explored if macrophage phenotypic state and function might alter upon treatment with selected NPs, and in turn, if macrophage phenotype alters the immunological response induced by selected NPs. In our study, we examined macrophage phenotype through multiplex cytokine analysis, focusing on 11 analytes indicative of macrophage polarization status. We utilized a specific mathematical formula, termed the macrophage polarization ratio (MPR), designed for this investigation. Results demonstrated that macrophages have an attraction for CN-functionalized NPs. In addition, the NPs have an impact on macrophage immunologic and phenotypic activity. The NPs induced high production of pro-inflammatory cytokines such IFN-ɣ, IL-1β, IL-12p70, IL-18, IL-2, IL-5, TNF-α, and IL-6. In summary, our study demonstrated the bacterial strain's is consistent in terms of replication homogeneity for both liquid culture and intracellularly, confirming M. tuberculosis's suitability for TB research with a reporter plasmid (pMV306hsp+Lux). Significantly, the CN-functionalized nanoparticles demonstrated noteworthy anti-mycobacterial efficacy. Data on macrophage viability highlighted the possible importance of a lower multiplicity of infection (MOI) for BMDMs. Recognizing the need for additional research, our findings indicated that CN-functionalized nanoparticles have a discernible influence on macrophage phenotype, specifically by increasing the expression of M1 markers. This comprehensive understanding highlights the promising role of CN-functionalized nanoparticles in shaping macrophage responses, implying avenues for targeted tuberculosis therapeutic interventions. By investigating the complex area of nanoparticles' impact on macrophage phenotype during TB treatment, this study filled a knowledge gap. This study expands our understanding of the dynamics of TB treatment and emphasizes the need for additional research to fully realize the promise of nanoparticle-mediated interventions in the treatment of TB. However, further research into the concentration-dependent anti-mycobacterial activity and transcriptomic response is required.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die veroorsakende middel van tuberkulose (TB), bly 'n groot kommer oor openbare gesondheid. Ten spyte van die beskikbaarheid van verskeie behandelingsregimes, is behandelingsmislukking algemeen, wat deels toegeskryf word aan die verskynsel van bakteriese persisters. Boonop kan herhaalde of langdurige behandelings moontlik dwelmweerstandige stamme van M. tuberculosis veroorsaak wat TB-beheer verder vererger. Nuwe benaderings tot TB-terapie is dus nodig. Om die intrasellulêre doodmaak van M. tuberculosis deur pro-inflammatoriese response (M1) te bevorder, is 'n belowende benadering die toepassing van nanopartikels (NP's), wat lipied-polimeer baster (LPN) insluit, om middels of immunomodulerende verbindings soos Curdlan (CN) na makrofage. Dit verteenwoordig 'n nuwe gasheergebaseerde terapeutiese benadering vir TB. Terwyl M1 gepolariseerde makrofage beskou word as die doeltreffendste in die beheer van M. tuberculosis infeksie, is dit getoon dat intrasellulêre basille 'n verskuiwing na die M2 fenotipe veroorsaak. Daar is geen bewyse dat die bekendstelling van NP's soos LPN wat met CN gekonjugeer is 'n verskuiwing in makrofaagfenotipe kan veroorsaak of dat die makrofaagpolarisasietoestand die anti-bakteriese aktiwiteit van NP's beïnvloed nie. In hierdie studie het ons eers begin om die anti-mikobakteriële doeltreffendheid van CN-gefunksionaliseerde NP's in vitro te evalueer, wat voorheen getoon het om belowend te wees in die inhibisie van mikobakteriese groei intrasellulêr en in vivo in 'n ouerstudie. Primêre selle afkomstig van die C57BL/6 muise beenmurg genoem beenmurg afgeleide makrofage (BMDMs) is vir hierdie studie gebruik. Ons het 'n verswakte M. tuberculosis H37Rv laboratoriumstam met 'n verslaggewerplasmied (pMV306hsp+Lux) gebruik om intydse monitering van luminessensie as 'n proxy vir die bakteriële lading moontlik te maak. Resultate van die intydse luminesensiemonitering het bevestig dat die CN-gefunksionaliseerde NP's 'n goeie anti-mikobakterie-middel is soos voorheen in die ouerstudie gedemonstreer. Ons het verder ondersoek of makrofaag fenotipiese toestand en funksie kan verander na behandeling met geselekteerde NP's, en op sy beurt, as makrofaag fenotipe die immunologiese respons wat deur geselekteerde NP's geïnduseer word, verander. In ons studie het ons makrofaagfenotipe deur middel van multipleks sitokienanalise ondersoek, met die fokus op 11 analiete wat 'n aanduiding is van makrofaagpolarisasiestatus. Ons het 'n spesifieke wiskundige formule gebruik, genaamd die makrofaagpolarisasieverhouding (MPR), ontwerp vir hierdie ondersoek. Resultate het getoon dat makrofage 'n aantrekkingskrag vir CNgefunksionaliseerde NPs het. Daarbenewens het die NP's 'n impak op makrofaag immunologiese en fenotipiese aktiwiteit. Die NP's het hoë produksie van pro-inflammatoriese sitokiene geïnduseer soos IFN-ɣ, IL-1β, IL12p70, IL-18, IL-2, IL-5, TNF-α en IL-6. Samevattend, ons studie het getoon dat die bakteriële stam konsekwent is in terme van replikasie-homogeniteit vir beide vloeibare kultuur en intrasellulêr, wat M. tuberculosis se geskiktheid vir TB-navorsing met 'n verslaggewerplasmied (pMV306hsp+Lux) bevestig. Dit is betekenisvol dat die CN-gefunksionaliseerde nanopartikels noemenswaardige antimikobakteriese doeltreffendheid getoon het. Data oor makrofagelewensvatbaarheid het die moontlike belangrikheid van 'n laer veelheid van infeksie (MOI) vir BMDM's uitgelig. Met die erkenning van die behoefte aan bykomende navorsing, het ons bevindinge aangedui dat CNgefunksionaliseerde nanopartikels 'n waarneembare invloed op makrofaag-fenotipe het, spesifiek deur die uitdrukking van M1-merkers te verhoog. Hierdie omvattende begrip beklemtoon die belowende rol van CNgefunksionaliseerde nanopartikels in die vorming van makrofage-reaksies, wat weë vir geteikende tuberkulose-terapeutiese intervensies impliseer. Deur die komplekse area van nanopartikels se impak op makrofaagfenotipe tydens TB-behandeling te ondersoek, het hierdie studie 'n kennisgaping gevul. Hierdie studie brei ons begrip van die dinamika van TBbehandeling uit en beklemtoon die behoefte aan bykomende navorsing om die belofte van nanopartikelgemedieerde intervensies in die behandeling van TB ten volle te verwesenlik. Verdere navorsing oor die konsentrasie-afhanklike anti-mikobakteriese aktiwiteit en transkriptomiese respons word egter vereis.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxvi, 97 pages : illustrationsen_ZA
dc.identifier.urihttps://scholar.sun.ac.za/handle/10019.1/130621en_ZA
dc.language.isoen_ZAen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMacrophagesen_ZA
dc.subject.lcshMycobacterium tuberculosisen_ZA
dc.subject.lcshDrug resistanceen_ZA
dc.subject.lcshNanoparticlesen_ZA
dc.subject.lcshDrug delivery systemsen_ZA
dc.titleThe influence of biomimetic nanoparticles on macrophage phenotype and their anti-mycobacterial efficacy on macrophages.en_ZA
dc.typeThesisen_ZA
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