Evaluation of synergistic hybrid nanoparticles for improved diagnosis and treatment of pancreatic cancerDoctoral thesis
Date of Examination:2023-07-06
Date of issue:2023-09-13
Advisor:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Susann Boretius
Referee:Prof. Dr. Ralf Dressel
Referee:Dr. Joanna Napp
Referee:Prof. Dr. Luis A. Pardo
Referee:Prof. Dr. Heidi Hahn
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EnglishPancreatic 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.
Keywords: molecular imaging; pancreatic cancer; nanoparticles; theranostic; gemcitabine; PDAC
Schlagwörter: molecular imaging; PDAC; gemcitabine; nanoparticles; pancreatic cancer; theranostic