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    Anti-Inflammatory Dendritic Polyglycerol Sulfate Nanoparticle: Biodistribution, Elimination, Cellular Localisation and Toxicopathology in Mice (2017)

    Art
    Hochschulschrift
    Autor
    Holzhausen, Cornelia (WE 12)
    Quelle
    Berlin, 2017 — X, 76 Seiten
    Sprache
    Englisch
    Verweise
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000105583
    Kontakt
    Institut für Tierpathologie

    Robert-von-Ostertag-Str. 15
    Gebäude 12
    14163 Berlin
    +49 30 838 62450

    Abstract / Zusammenfassung

    The prefix “nano” refers to any material smaller than 100 nm in one

    dimension. Materials with all dimensions in the nanoscale are defined as

    nanoparticles (NPs). At this dimension quantum effects unfold and

    nanomaterials (NMs) show an enhanced reactivity due to their high

    area-to-mass ratio. Many opportunities for innovations in industry and

    biomedicine originate from the unique features of NMs. In biomedicine

    their applications include medical devices, in vitro and in vivo

    diagnostics, drug delivery and therapeutics. A promising NP for

    different biomedical applications, in which the most prominent are the

    diagnosis and therapy of inflammation, is the dendritic polyglycerol

    sulfate (dPGS). However, the unique features of NMs can also lead to

    unforeseeable behaviour and may bear safety risks.

    Problems in risk management of NMs are in particular the missing

    consensus and availability of methods adequate for safety testing.

    Furthermore, to date specific regulatory guidance has been released for

    only a few NMs. The vast majority of NMs are regulated in preclinical

    approval like “regular” small drugs in accordance with the guidance

    document M3(R2) of the International Council for Harmonisation of

    Technical Requirements for Pharmaceuticals for Human Use. With reference

    to M3(R2), preclinical drug approval includes pharmacology,

    toxicokinetics and pharmacokinetics as well as toxicity studies. Yet

    further parameters, related to the unique features of NMs, such as

    particle size or aggregation effects, may be needed for risk assessment.

    Moreover, standard tests can interfere with NMs and require

    verification.

    The study presented here surveys the kinetics as well as the acute and

    subacute toxicopathology of dPGS and thus contributes to providing a

    basis for further research with a view towards drug approval. In

    previous in vivo efficacy studies fluorescently labelled dPGS showed no

    obvious acute adverse effects. The assessment of dPGS for biomedical

    applicability has so far been promising, but no systematically surveyed

    data regarding the distribution, elimination and histopathology of

    healthy animals, as common determinants in preclinical safety

    assessment, were available. Furthermore, fluorescent labelling is

    thought to alter NP properties leading to a different biodistribution,

    and hence requires verification. In addition, fluorescent labelling does

    not allow for histopathological evaluation. Techniques feasible to trace

    “soft” NPs such as dPGS in vivo are, however, limited. Due to their

    small size, all NPs escape the wavelength of light and require labelling

    for light microscopy, while the low atomic number of “soft” NPs also

    rules out electron microscopy. There are innovative methods, such as

    Raman microspectroscopy, which do not require labelling and allow NPs to

    be localised in cells and enable cellular alterations to be evaluated,

    but have the disadvantage of being technically demanding and not widely

    available. An alternative to labelling with fluorophores is labelling

    with radioisotopes. Provided the isotope is well integrated, the

    chemical structure of the NP is preserved. The radiation can be recorded

    by whole body/organ autoradiography (WBA/WOA), but can also be used for

    quantitative kinetic studies employing quantitative whole body/organ

    autoradioluminography (QWBA/QWOA) or liquid scintillation counting

    (LSC). Yet, these techniques only provide data on tissue level. To

    detect radioactively labelled NPs in cells, the light microscopic

    autoradiography (LMA) was established in this study. LMA also allows for

    concurrent histopathological analysis.

    The radioisotope 35S was incorporated into the dPGS NP

    specifically for this study. This isotope was particularly suitable

    because sulfur is a constituent of dPGS. It should be noted that amino

    functions were integrated for easy conjugation with a drug or dye. The

    resulting radioactive dPG35S amine was administered i.v. or

    s.c. to healthy mice. The mice were sacrificed but not exsanguinated at

    different time points following application, and samples were collected

    for LSC or autoradiography (WOA, QWOA, and LMA). The dPG35S

    amine concentration in liver and spleen increased up to 5 and 21 days

    following i.v. application respectively. Evaluation of tissue sections

    with LMA localised dPG35S amine in the Kupffer cells of the

    liver and in the red pulp of the spleen 24 hours, 5 and 21 days post

    dose. In other organs such as the kidney, lung, intestine, testes, and

    brain the overall dPG35S amine concentration decreased over

    time. Only low concentrations were measured in the testes and marginal

    concentrations in the brain, suggesting a tight blood-tissue barrier for

    this NP. Furthermore, dPG35S amine was found in the faeces

    and at early time points in the urine. Taken together these data do

    indeed suggest a partial elimination via the liver and kidney, but apart

    from that dPG35S amine is retained in the Mononuclear

    Phagocyte System (MPS). Particles that are not readily degraded in

    macrophages become sequestered, as confirmed by LMA for

    dPG35S amine. The long apparent terminal blood half-life of

    dPG35S amine, exceeding 12 days, also indicates retention.

    Apart from the delayed onset, the distribution of this NP to the organs

    after s.c. application was very similar to its i.v. application.

    In this study neither the clinical evaluation during the experiment nor

    the gross and histopathological analysis of the examined tissue showed

    any adverse effects. Thus, from a pathomorphological point of view,

    there was no evidence that would impede future investigations of dPGS

    for biomedical usage. Accumulation of dPGS in MPS cells, which is known

    for other charged NPs as well and depends on the protein corona of the

    NP, however, always bears the risk of toxicity and hinders an

    application as a therapeutic or diagnostic agent. In addition, retained

    NPs may interfere with diagnostic imaging. The retention of dPGS in MPS

    cells will have to be addressed using long-term and repeated-dose

    toxicity testing as well as in any further attempts to develop dPGS for

    biomedical applications.