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): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000105583

Kontakt

Institut für Tierpathologie

Robert-von-Ostertag-Str. 15
Gebäude 12
14163 Berlin
Tel.+49 30 838 62450 Fax.+49 30 838 62522

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.