RésuméCancer is the second leading cause of mortality worldwide, responsible for an estimated 9.6 million deaths in 2018. Currently, the conventional therapeutic approaches are surgical removal of the tumor, chemotherapy and/or radiotherapy. However, despite the recent advances in cancer treatment, a significant number of patients still experience tumor recurrence and have serious side effects due to the damage caused to healthy tissues. Therefore, there is a need to further improve the current treatment modalities, or develop new therapeutic strategies, which enhance the cancer cell killing, while sparing the healthy tissues. Thanks to their unique optical and physicochemical properties, gold nanoparticles (AuNPs) have emerged in nanomedicine as promising contrast agents, drug delivery vehicles, photo-thermal agents and radiosensitizers. In order to maximize the concentration of AuNPs into the tumor site, surface modifications with targeting moieties are typically applied onto the AuNP surface.
In this thesis, we used 5 nm AuNPs coated with organic poly-allylamine (AuNPs-PAA) and conjugated to Cetuximab (Ctxb), a commercially available antibody targeting the epidermal growth factor receptor (EGFR). EGFR is overexpressed in numerous cancer types, such as colorectal, head- and neck cancers, breast cancer and prostate cancer. Previously, it has been shown that the resulting AuNPs-PAA-Ctxb are able to selectively target EGFR-overexpressing cancer cells in vitro and in vivo, and enhance the efficiency of proton therapy in EGFR-overexpressing cancer cells. In nanomedicine, intravenous administration of AuNPs-PAA-Ctxb would be the most realistic exposure scenario. However, following systemic administration, it is inevitable that healthy cells and tissues will be exposed to the AuNPs-PAA-Ctxb as well. The first cells that would encounter the injected AuNPs-PAA-Ctxb are the endothelial cells, lining the inner walls of the vasculature. Furthermore, the hepatobiliary system and the renal system are potential elimination routes to remove AuNPs-PAA-Ctxb from the body. Therefore, we investigated the cytotoxic effects of AuNPs-PAA and AuNPs-PAA-Ctxb in human microvascular endothelial cells, liver cells and kidney cells. After exposure, AuNPs-PAA-Ctxb were internalized in all cell types and were present in intracellular vesicles. Interestingly, the cellular EGFR expression profile was not a prognostic factor to predict the sensitivity of the cells to the effects of AuNPs-PAA-Ctxb. Instead, we reported that AuNPs-PAA and AuNPs-PAA-Ctxb caused mitochondrial dysfunction and significantly suppressed the activity of the antioxidant enzymes, thioredoxin reductase (TrxR) and glutathione reductase (GR), finally resulting in apoptotic cell death. The role of oxidative stress was highlighted by the protective effects of the antioxidant N-acetyl L-cysteine, which prevented mitochondrial dysfunction and considerably reduced apoptosis. Finally, we evidenced that the basal TrxR activity, the extent of TrxR inhibition and the mitochondrial dysfunction are strongly correlated with the sensitivity of the normal cells to AuNPs-PAA and AuNPs-PAA-Ctxb.
Since we observed cytotoxic effects of AuNPs-PAA-Ctxb in normal cells in vitro, we assessed, the pharmacokinetics, biodistribution and toxicity of the AuNPs-PAA-Ctxb after a single intravenous injection in healthy mice. The AuNPs-PAA-Ctxb were rapidly cleared from the blood circulation, followed by their accumulation and long-term retention in the liver and spleen. However, we did not observe significant morphological changes in the liver, kidney, spleen and lungs up to four weeks post-injection. In addition, there was only a minimal and transient increase in a limited number of serum markers related to the immune response, endothelial activation and liver toxicity.
Finally, since the AuNPs-PAA-Ctxb clearly affected some biological systems involved in the antioxidant defense and cell survival, AuNPs-PAA-Ctxb could predispose cancer cells to apoptosis after exposure to ionizing radiation. The radiosensitizing effects of AuNPs are usually investigated considering external beam radiotherapy (EBRT). However, there are only a limited number of studies investigating the use of AuNPs as radiosensitizing agents in combination with radionuclides. Therefore, in the last part, we radiolabeled the AuNPs-PAA-Ctxb to the theragnostic radionuclide, 177Lu, using bifunctional chelators conjugated to Ctxb. The bifunctional chelator diethylenetriaminepentaacetic acid (DTPA) was preferred because of the fast radiolabeling reaction under mild conditions and the minimal impact on the Ctxb binding capacity. 177Lu-DTPA-Ctxb-PAA-AuNPs preserved their ability to recognize EGFR and exhibited a more extensive binding and internalization in cancer cells compared to 177Lu-DTPA-Ctxb. As a result, together with the biological inhibition, 177Lu-DTPA-Ctxb-PAA-AuNPs could have the potential to increase the efficacy of targeted radionuclide therapy compared to 177Lu-DTPA-Ctxb.
In order to assess the radiosensitizing properties of the 177Lu-DTPA-Ctxb-PAA-AuNPs in targeted radionuclide therapy, future experiments will be conducted, assessing the cell viability and clonogenic potential of various cancer cell types with different EGFR expression and TrxR activity profiles, after exposure to 177Lu-DTPA-Ctxb-PAA-AuNPs or 177Lu-DTPA-Ctxb. In addition, 177Lu-radiolabeling of AuNPs-PAA-Ctxb will enable the real-time imaging of the biodistribution and elimination of the nanoconjugate in mice, bearing EGFR-positive and EGFR-negative tumor xenografts. Finally, in vivo preclinical therapy experiments will inform whether 177Lu-DTPA-Ctxb-PAA-AuNPs bear superior tumor killing capacity.
Altogether, 177Lu-DTPA-Ctxb-PAA-AuNPs is a potentially superior radiopharmaceutical as compared to 177Lu-DTPA-Ctxb, because of the (I) mitochondrial dysfunction and suppression of the antioxidant system, (II) the enhanced cancer cell internalization and (III) the delivery of multiple 177Lu radionuclides per AuNP. However, the rapid sequestration and long-term retention of the AuNPs-PAA-Ctxb in the liver and spleen after intravenous administration might restrict the tumor accumulation and thus the therapeutic potential of 177Lu-DTPA-Ctxb-PAA-AuNP.
|la date de réponse||2 sept. 2020|
|Sponsors||Fund for Research Training in Industry and Agriculture (FRIA)|
|Superviseur||Stephane Lucas (Promoteur), Carine MICHIELS (Copromoteur), An Aerts (Copromoteur), BERNARD MASEREEL (Président), Karen Van Hoecke (Jury), Sarah Baatout (Jury) & Nadine Millot (Jury)|