Surface-functionalized nanomaterials for a drug-delivery system in cancer immunotherapy
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Abstract
In cancer therapy there is a vast demand on novel cell type-specific drug delivery systems. High tunable engineered nanomaterials bear many characteristics that make them attractive as a drug carrier for improved immunotherapy. This thesis aims to disclose the biological interaction with as well as the uptake by primary human immune cells of chemically modified polystyrene nanoparticles and biologically functionalized hydroxyethyl starch nanocapsules.
In a first approach, polystyrene nanoparticles were studied as a model system for investigating the influence of chemical surface modifications on the uptake of nanoparticles by human dendritic cells and their immunogenicity. Fluorescently labeled sulfonate- and phosphonate-functionalized polystyrene nanoparticles were found to be taken up by human monocyte-derived immature and mature dendritic cells as measured by flow cytometry. Confocal laser scanning microscopy ascertained the intracellular nanoparticle uptake. The nanoparticle influence on dendritic cell maturation was addressed by the use of human immature monocyte-derived dendritic cells that can differentiate into mature dendritic cells upon activation. Both nanoparticles induced enhanced maturation of immature dendritic cells as depicted by upregulation of dendritic cell maturation markers as well as co-stimulatory molecules accompanied with elevated amounts of cytokines associated with dendritic cell activation compared to untreated dendritic cells. Sulfonate- as well as phosphonate-functionalized nanoparticles increased the T cell stimulatory potential of immature dendritic cells indicating a T helper type 1 cell response.
In a second approach, biodegradable hydroxyethyl starch nanocapsules were surface-functionalized with the cytokine IL-2 and their interaction with and uptake by IL-2 receptor-bearing T cells was assessed. The biological functionality of IL-2 linked to the capsule surface was confirmed on IL-2-dependent growing murine CTLL-2 cells. In order to investigate the uptake of IL-2-functionalized nanocapsules, primary human CD4+ T cells were isolated from peripheral blood and activated for increased CD25 expression, which represents the IL-2 receptor alpha subunit and is decisive for internalization of the high affinity IL-2 receptor. IL-2-functionalized hydroxyethyl starch nanocapsules showed a significant uptake by CD4+CD25high T cells compared to control capsules. Along capsule uptake IL-2-induced proliferation of CD4+CD25high T cells was assessed with a proliferation dye by flow cytometry. In order to analyze the impact of different amounts of IL-2 linked to the nanocapsules, hydroxyethyl starch nanocapsules were surface-functionalized with half the amount of IL-2. Reduced amounts of IL-2 resulted in diminished CD4+CD25high T cell proliferation and impaired uptake efficiency by CD4+CD25high T cells. Moreover, IL-2-functionalized hydroxyethyl starch nanocapsules were intravenously injected in mice and their biodistribution in spleen, liver and lymph nodes was measured. Elevated levels of nanocapsule-positive CD4+CD25+ T cells isolated from the lymph nodes were detected compared to control mice.
In summary, in this study polystyrene nanoparticles were analyzed for their chemical surface modification, whereas hydroxyethyl starch nanocapsules were even ligand-functionalized for a cell type-specific uptake. Studying IL-2-functionalized hydroxyethyl starch nanocapsules revealed a cell type-specific uptake by CD4+CD25high T cells. This study provides substantial findings for the future development of IL-2-functionalized nanocapsules as a drug delivery system for CD4+CD25high T cells in cancer immunotherapy.