Manipulation of macrophage phagocytosis - development of an endogenous delivery system

Visser, Johan Georg (2020-04)

Thesis (PhD)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: The need to administer more potent antimicrobial drugs is supported by the ever-increasing incidence of multidrug resistance. Given the (necessary) higher toxicity of these drugs, administration into host circulation comes at a high risk to the patient. Drug delivery systems that are capable of more localized drug deposition, could limit host exposure. Here we propose the use of an autologous delivery system to shuttle drugs through circulation to protect the host from premature drug exposure. Our approach encompassed a multidisciplinary method to include physiology and microbiology. From the physiology side, macrophages exhibit great capacity to transverse endothelial barriers during the inflammatory process. From the microbiology side, micro-organisms have evolved to evade the immune system by harboring within these macrophages to later induce their own expulsion for dissemination. The work presented here describes how we have utilized the pore forming and actin polymerising ability of the Listeria monocytogenes effectors, listeriolysin-O and actin assembly-inducing protein, to produce a novel drug delivery system: the synthetic microbe. Firstly, we synthesised these effectors by using a GFP-linked heterologous expression and purification system, with which we were able to produce effectors at a greater yield than previously reported. In vitro experiments further confirmed appropriate activity of synthesised proteins and finally, coating of these effector proteins onto polystyrene beads induced their expulsion from carrier macrophages. Furthermore, drug cargo expulsion did not result in lysis of the carrier cells, suggesting that macrophages could contribute to resolution of damage at target areas once cargo is released. In our opinion, this multidisciplinary approach may hold the solution to effective, controlled drug delivery.

AFRIKAANSE OPSOMMING: Die noodsaaklikheid om kragtiger antimikrobiese middels toe te dien, word beklemtoon deur die toenemende voorkoms van veelvuldige middel weerstandigheid. Gegewe die (noodsaaklike) hoër toksisiteit van hierdie medisyne, hou die sistemiese toediening daarvan 'n groot risiko vir die pasiënt in. Medisyne-afleweringstelsels wat meer gelokaliseer kan word, kan blootstelling van die gasheer beperk. Hier word voorgestel dat 'n outoloë afleweringstelsel gebruik word om dwelms deur die sirkulasie te vervoer, wat die gasheer teen voortydige blootstelling aan medisyne beskerm. Ons het 'n multidissiplinêre benadering ingespan wat beide fisiologie en mikrobiologie insluit. Van die fisiologiese kant af besit makrofage die vermoë om dwarsbrekings deur die endoteel te maak gedurende die inflammatoriese proses. Van die mikrobiologiese kant af het mikroörganismes ontwikkel om die immuunstelsel te ontduik deur binne hierdie makrofage weg te kruip en later hul eie uitsetting vir verspreiding te bewerkstellig. Die werk wat hier aangebied word, beskryf hoe ons die porievorming en aktienpolimerisasie-vermoë van die Listeria monocytogenes-effektore listeriolysien-O en aktien-samestellende induserende proteïen gebruik het om 'n nuwe medisyne-afleweringstelsel te vervaardig: die sintetiese mikroörganisme. Eerstens het ons hierdie effektore gesintetiseer deur gebruik te maak van 'n GFP-gekoppelde heteroloë uitdrukking- en suiweringstelsel, waarmee ons effektore met 'n groter opbrengs kon produseer as wat voorheen gerapporteer is. In vitro-resultate het die toepaslike aktiwiteit van gesintetiseerde proteïene verder bevestig. Laastens het die bedekking van hierdie effektorproteïene op polistireenkrale hul uitsetting uit draer-makrofage veroorsaak. Verder het die uitsetting van geneesmiddelvragte nie gelei tot lise van die draerselle nie, wat daarop dui dat makrofage kan bydra tot die genesing van skade in die teikengebiede nadat die vrag vrygestel is. Na ons mening kan hierdie multidissiplinêre benadering die oplossing vir effektiewe, beheerde medisyne-aflewering inhou.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/107782
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