| dc.description.abstracteng | Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis with no effective treatment
options. Several factors contribute to its lethality, including the usually late diagnosis in the already
advanced stage and the limited effectiveness of current chemotherapeutic drugs, resulting in low
median patient survival rates. Furthermore, the development of chemoresistance shortly after
treatment initiation contributes to the failure to increase the survival rates of patients. Despite
significant investment in time and resources over years, the development of satisfactory diagnostic
tools and efficient treatment for PDAC has to date been unsuccessful. Thus, there is an urgent need
for innovative more effective and safe therapies. This PhD research project aims to preclinically
evaluate the effectiveness of a novel class of nanocarriers, inorganic-organic hybrid nanoparticles (IOHNPs) for selective delivery of extraordinarily high concentrations of cytostatic drugs to the tumour and
metastatic sites in murine and human PDAC mouse models.
To achieve optimal anti-tumour efficacy whilst minimizing side effects and systemic toxicity, a
comprehensive preclinical evaluation of the IOH-NPs was conducted in vitro, in vivo, and ex vivo,
employing various advanced fluorescence imaging techniques. The IOH-NP portfolio encompassed two
primary types of NPs, differing in their metal core: zirconium- and gadolinium-based NPs. Additionally,
for effective tracking and visualization, IOH-NPs were labelled with fluorescence dyes, DUT549 for in
vitro and DUT647 for in vivo fluorescence imaging. Notably, synthesis of IOH-NPs resulted in a high
payload of up to 60-80% of chemotherapeutic agents (e.g. gemcitabine). Among the chemotherapies
loaded in the IOH-NPs, gemcitabine was of particular interest as the current first-line treatment for
PDAC. Furthermore, a combination of chemotherapeutic agents (e.g. SN38 or 5-FU) was incorporated
into the IOH-NPs, to further expand their potential therapeutic capabilities in PDAC. Lastly, glucose
(Glu) coating was used to improve the cellular uptake based on the high metabolic activity of PDAC
cells and cetuximab (CTX) functionalization was added to improve targeting to PDAC cells, a variety of
which overexpress hEGFR on their surface.
Using non-active reference IOH-NPs, we showed in vitro that all the IOH-NPs were taken up by PDAC
cells (murine KPC, Panc02, and human AsPC1, Capan-1, and BxPC3). The best uptake was obtained with
the zirconium-based IOH-NPs, while the gadolinium-based Core-Shell NPs were taken up less. Out of
the glucose coated and CTX functionalized zirconium-based NPs, the non-coated and nonfunctionalized NPs showed superior uptake compared to other formulations. Neither glucose coating,
nor functionalization with the targeting ligand CTX, was able to improve NP cell uptake in vitro.
Of the IOH-NPs tested in vitro, the best cytotoxic efficacy was obtained with the zirconium-based Gemtriphosphate NPs. The uptake of Gem-NPs was independent of the activity of the human equilibrative
nucleoside transporter (hENT1), responsible for gemcitabine transport into cells and for developing
chemoresistance. These results led us to use gemcitabine, as the cytostatic drug of choice for
preclinical treatment studies. It is noteworthy that gemcitabine converts to its active form, after its
entrance in the cell, via triphosphorylation. By using the monophosphate form, the first stage of
phosphorylation (via deoxycytidine kinase) is omitted, potentially enhancing the bioavailability of the
drug. By using the triphosphate form, all stages of phosphorylation are bypassed, thereby providing
already the active form that competes with dCTP for incorporation into DNA and leading to an
inhibition of DNA synthesis. This is in accordance with our finding that Gem-triphosphate NPs showed
higher efficacy in vitro than gemcitabine-monophosphate. However, for cost reasons, only the
monophosphate form of gemcitabine was used for subsequent in vivo studies. Therefore, zirconiumbased gemcitabine-monophosphate loaded IOH-NP (Gem-M-NPs) were selected for the in vivo efficacy
studies.11
Here two different mouse models were used: The Panc02 orthotopic syngeneic PDAC mouse model
(syngeneic C57BL/6 mouse model transplanted with Panc02 murine cell line) and the AsPC1-PDAC
mouse model (xenograft NMRI/NMRI Fox1nu/nu transplanted with human AsPC1 cell line). In vivo,
intraperitoneally (I.P.) applied reference NPs selectively accumulated within tumour nodules, while
intravenous (I.V.) administration resulted in accumulation not only in the tumour but also in the liver.
Due to low liver uptake, I.P. administration was chosen for subsequent preclinical treatment efficacy
studies.
Gem-M-NPs showed the best efficacy in reducing tumour growth in the Panc02 orthotopic syngeneic
PDAC mouse model, while Gem-M-NPs, functionalized with cetuximab, (Gem-CTX-NPs) demonstrated
the strongest anti-tumour effect in the AsPC1-PDAC mouse model. Treatment with Gem-CTX-NPs
resulted in smaller tumour sizes measured by caliper compared to the untreated control group. The
anti-tumour efficacy was assessed by the appearance of apoptotic areas within AsPC1 and Panc02
tumours after performing tunel assay in response to the treatment, where AsPC1 tumours treated with
Gem-CTX-NPs and Panc02 tumours treated with Gem-M-NPs resulted in the most apoptotic areas,
respectively.
Fluorescent imaging combined with fluorescently labelled IOH-NP confirmed the uptake of IOH-NP in
tumour cells. Gem-M-NPs highly accumulated in tumour lesions, most likely as intact IOH-NP,
protecting gemcitabine during the delivery process, with almost no liver trapping when given
intraperitoneally. Importantly, both Gem-M-NPs and Gem-CTX-NPs showed higher anti-tumour
efficacy compared to the free drug.
Imaging with light sheet fluorescence microscopy (LSFM) proved the high delivery of NPs in the
tumour, mainly in the periphery, verifying the integrity and enlightening the distribution of the NPs
within the tumours. Spectrum analysis of histological slides confirmed that the NPs reached the
tumours, by measuring the fluorescence dye the NPs are labelled with. Additionally, Gem-CTX-NPs
were accumulated in areas with EGFR overexpressing tumour cells, resulting in successful targeting of
tumour cells with NPs. Comprehensive preclinical evaluation provides valuable insights into the
therapeutic effects of different IOH-NPs, especially of gemcitabine-containing formulations, facilitating
further optimization of IOH-NPs for targeted treatment strategies for PDAC. | de |