Browsing by Author "Visser, Johan Georg"

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    Development of a transendothelial shuttle by macrophage modification
    (Stellenbosch : Stellenbosch University, 2016-12) Visser, Johan Georg; Smith, Carine; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Background: Targeted stem cell delivery via macrophage modification is a novel and relatively non-invasive therapeutic intervention. Monocytes circulate through the vasculature and infiltrate damaged tissue in response to chemotactic signalling. Here they differentiate into functionally different macrophage phenotypes - the classically activated pro-inflammatory (M1) or alternatively activated anti-inflammatory (M2) phenotypes. The M1 macrophages are able to cross endothelial barriers, while the M2 anti-inflammatory macrophages are unable to transverse endothelium and instead remain tissue associated. The focus of our research was to produce M1 macrophages in vitro that could transverse endothelium while carrying engulfed stem cells, in order to deliver more stem cells in a relatively short time, to any injured tissue to facilitate recovery. Methods: Primary isolated monocytes were cultured with Granulocyte Monocyte Colony-Stimulating Factor, Lipopolysaccharide and Interferon gamma for 6 days to pre-differentiate them into M1 macrophages. Cells were treated with a Wortmannin-Concanamycin A-Chloroquine cocktail to achieve phagosome maturation arrest and thus preserve ingested cells in a viable state. Preservation of engulfed stem cells (simulated with fluorescent latex beads covalently labelled with IgG antibody) was qualitatively and quantitatively determined by flow cytometry and live cell imaging, respectively. Bead-containing macrophages were co-cultured with HUVEC cells in a Transwell system, and exposed to Monocyte Chemoattractant Protein 1 (added to bottom well) to determine migration capacity. Results: Monocytes were differentiated into M1 classically activated macrophages. The majority of these cells (68.67±3.51%) were able to engulf opsonised beads after successful induction of phagosome maturation arrest – a capacity similar to that of untreated cells (61.19±4.68%). Ingested beads were preserved within macrophages for the duration of our protocol (2 hours), determined by retained red antibody signal on beads and perturbed phagosome acidification. 72.86±16.0 phagosome maturation arrested macrophages were able to transverse a HUVEC coated membrane with 8 μm diameter pores (simulated endothelial layer), while only 70.14±12.6 cells per well migrated when carrying a bead cargo. Conclusion: A delivery system capable of engulfing, preserving and delivering cargo was successfully induced. Further optimisation of this technique could lead to translation into a novel method for delivery of stem cells with regard to regenerative medicine and may even be used as a drug delivery system for the treatment of various malignancies.
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    Harnessing Macrophages for Controlled-Release Drug Delivery : lessons From Microbes
    (Frontiers Media, 2019) Visser, Johan Georg; Du Preez Van Staden, Anton; Smith, Carine
    With the effectiveness of therapeutic agents ever decreasing and the increased incidence of multi-drug resistant pathogens, there is a clear need for administration of more potent, potentially more toxic, drugs. Alternatively, biopharmaceuticals may hold potential but require specialized protection from premature in vivo degradation. Thus, a paralleled need for specialized drug delivery systems has arisen. Although cell-mediated drug delivery is not a completely novel concept, the few applications described to date are not yet ready for in vivo application, for various reasons such as drug-induced carrier cell death, limited control over the site and timing of drug release and/or drug degradation by the host immune system. Here, we present our hypothesis for a new drug delivery system, which aims to negate these limitations. We propose transport of nanoparticle-encapsulated drugs inside autologous macrophages polarized to M1 phenotype for high mobility and treated to induce transient phagosome maturation arrest. In addition, we propose a significant shift of existing paradigms in the study of host-microbe interactions, in order to study microbial host immune evasion and dissemination patterns for their therapeutic utilization in the context of drug delivery. We describe a system in which microbial strategies may be adopted to facilitate absolute control over drug delivery, and without sacrificing the host carrier cells. We provide a comprehensive summary of the lessons we can learn from microbes in the context of drug delivery and discuss their feasibility for in vivo therapeutic application. We then describe our proposed “synthetic microbe drug delivery system” in detail. In our opinion, this multidisciplinary approach may hold the solution to effective, controlled drug delivery.
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    Manipulation of macrophage phagocytosis - development of an endogenous delivery system
    (Stellenbosch : Stellenbosch University, 2020-03) Visser, Johan Georg; Smith, Carine; Van Staden, Anton du Preez; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    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.