Publikationsdatenbank

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): https://refubium.fu-berlin.de/handle/fub188/5593

Kontakt

Institut für Tierpathologie

Robert-von-Ostertag-Str. 15
14163 Berlin
+49 30 838 62450
pathologie@vetmed.fu-berlin.de

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.