Hepatocellular carcinoma (HCC) is the most common type of liver cancer and accounts for 90% of all cases worldwide. It is estimated that more than one million individuals will be affected by liver cancer by 2025, making it a global health challenge. The staging and prognostic assessment of HCC is crucial for patients’ management and the tumor is divided into stages, each of them related to different treatment options. To date, HCC early diagnosis is achievable through radiological and biopsy approaches. Despite all the diagnostic efforts, about 50% of all cases are diagnosed for other reasons and when the tumor is already at the late stage dramatically limiting treatment options. Thus, there is the need to discover new tumor-associated antigens with clinical significance for the early diagnosis and the development of targeted therapies. In this context, glypican-3 (GPC-3) protein is an heparan sulphate proteoglycan that is not detected in healthy adult liver but is overexpressed in 70-80% of patients suggesting its involvement in the malignant transformation of HCC. Moreover, in response to constraints in current HCC management, nanomedicine has tremendous potential applications. The implementation of multifunctional nanoparticles (NPs) that combine active cancer targeting, imaging, and therapeutic functionalities into a single platform (theranostics) represents the future. As a result, the proposed approach was the development of a novel theranostic nanosystem composed of: an anti-GPC-3 antibody for the active HCC targeting, a polymeric poly(lactic-co-glycolic acid) (PLGA)-based nanocarrier, and gold nanoparticles (GNPs) as imaging and therapeutic agents. Primarily, GPC-3 protein expression was assessed in different cancer cell lines (GPC-3 positive and negative), demonstrating the utility of GPC-3 as a suitable target and TAA for HCC. Hence, to obtain a specific custom-made anti-GPC-3 antibody, the production of mouse IgG monoclonal antibodies was implemented leading to the selection of D8 antibody being the most promising in terms of GPC-3 recognition and production yield. Following the characterization of D8 antibody, that revealed comparable performances as compared to clinical-used GC33 antibody, it was selected to be functionalized onto the NPs surface. PLGA nanocarrier was synthesised and in vitro characterized demonstrating excellent physiochemical properties. PLGA-NPs showed an average diameter of 250 nm, spherical nanocapsular morphology, thermal, pH and over time stability, absent cytotoxicity, and great ability to be internalized into HCC cells. As theranostic element, hydrophilic 14-15 nanometer-sized GNPs were chemically produced and characterized, and therefore encapsulated inside the PLGA-NPs core (hybrid NPs). The encapsulation process was confirmed by means of several techniques, and the gold amount quantified for in vivo applications. To test the contrasting ability, hybrid NPs were analyzed by X-ray Computed Tomography showing promising results in the potential to be undoubtedly distinguished from surroundings especially when resuspended in a membrane matrix mimicking the biological environment. Finally, the hybrid NPs were covalently functionalized with D8 antibody and in vivo and ex vivo investigated. When intravenously (IV) injected in HCC xenograft mouse model following the biodistribution for 48h timepoint, targeted and untargeted hybrid NPs mainly accumulated in elimination organs (liver and spleen) to a larger extent as compared to GNPs alone. However, D8 hybrid NPs exhibited a higher accumulation and retention in the tumor mass corroborating the benefit of an active cancer targeting approach. Despite the encouraging results obtained, the proposed system should be better characterized and investigated. Nevertheless, it lays the groundwork for potential in vivo application in clinical setting.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and accounts for 90% of all cases worldwide. It is estimated that more than one million individuals will be affected by liver cancer by 2025, making it a global health challenge. The staging and prognostic assessment of HCC is crucial for patients’ management and the tumor is divided into stages, each of them related to different treatment options. To date, HCC early diagnosis is achievable through radiological and biopsy approaches. Despite all the diagnostic efforts, about 50% of all cases are diagnosed for other reasons and when the tumor is already at the late stage dramatically limiting treatment options. Thus, there is the need to discover new tumor-associated antigens with clinical significance for the early diagnosis and the development of targeted therapies. In this context, glypican-3 (GPC-3) protein is an heparan sulphate proteoglycan that is not detected in healthy adult liver but is overexpressed in 70-80% of patients suggesting its involvement in the malignant transformation of HCC. Moreover, in response to constraints in current HCC management, nanomedicine has tremendous potential applications. The implementation of multifunctional nanoparticles (NPs) that combine active cancer targeting, imaging, and therapeutic functionalities into a single platform (theranostics) represents the future. As a result, the proposed approach was the development of a novel theranostic nanosystem composed of: an anti-GPC-3 antibody for the active HCC targeting, a polymeric poly(lactic-co-glycolic acid) (PLGA)-based nanocarrier, and gold nanoparticles (GNPs) as imaging and therapeutic agents. Primarily, GPC-3 protein expression was assessed in different cancer cell lines (GPC-3 positive and negative), demonstrating the utility of GPC-3 as a suitable target and TAA for HCC. Hence, to obtain a specific custom-made anti-GPC-3 antibody, the production of mouse IgG monoclonal antibodies was implemented leading to the selection of D8 antibody being the most promising in terms of GPC-3 recognition and production yield. Following the characterization of D8 antibody, that revealed comparable performances as compared to clinical-used GC33 antibody, it was selected to be functionalized onto the NPs surface. PLGA nanocarrier was synthesised and in vitro characterized demonstrating excellent physiochemical properties. PLGA-NPs showed an average diameter of 250 nm, spherical nanocapsular morphology, thermal, pH and over time stability, absent cytotoxicity, and great ability to be internalized into HCC cells. As theranostic element, hydrophilic 14-15 nanometer-sized GNPs were chemically produced and characterized, and therefore encapsulated inside the PLGA-NPs core (hybrid NPs). The encapsulation process was confirmed by means of several techniques, and the gold amount quantified for in vivo applications. To test the contrasting ability, hybrid NPs were analyzed by X-ray Computed Tomography showing promising results in the potential to be undoubtedly distinguished from surroundings especially when resuspended in a membrane matrix mimicking the biological environment. Finally, the hybrid NPs were covalently functionalized with D8 antibody and in vivo and ex vivo investigated. When intravenously (IV) injected in HCC xenograft mouse model following the biodistribution for 48h timepoint, targeted and untargeted hybrid NPs mainly accumulated in elimination organs (liver and spleen) to a larger extent as compared to GNPs alone. However, D8 hybrid NPs exhibited a higher accumulation and retention in the tumor mass corroborating the benefit of an active cancer targeting approach. Despite the encouraging results obtained, the proposed system should be better characterized and investigated. Nevertheless, it lays the groundwork for potential in vivo application in clinical setting.

Development of a Novel Hybrid Nanoplatform for Theranostic Applications in Hepatocellular Carcinoma (HCC) / Steffè, Aharon. - (2023 May 19).

Development of a Novel Hybrid Nanoplatform for Theranostic Applications in Hepatocellular Carcinoma (HCC)

STEFFÈ, AHARON
2023-05-19

Abstract

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and accounts for 90% of all cases worldwide. It is estimated that more than one million individuals will be affected by liver cancer by 2025, making it a global health challenge. The staging and prognostic assessment of HCC is crucial for patients’ management and the tumor is divided into stages, each of them related to different treatment options. To date, HCC early diagnosis is achievable through radiological and biopsy approaches. Despite all the diagnostic efforts, about 50% of all cases are diagnosed for other reasons and when the tumor is already at the late stage dramatically limiting treatment options. Thus, there is the need to discover new tumor-associated antigens with clinical significance for the early diagnosis and the development of targeted therapies. In this context, glypican-3 (GPC-3) protein is an heparan sulphate proteoglycan that is not detected in healthy adult liver but is overexpressed in 70-80% of patients suggesting its involvement in the malignant transformation of HCC. Moreover, in response to constraints in current HCC management, nanomedicine has tremendous potential applications. The implementation of multifunctional nanoparticles (NPs) that combine active cancer targeting, imaging, and therapeutic functionalities into a single platform (theranostics) represents the future. As a result, the proposed approach was the development of a novel theranostic nanosystem composed of: an anti-GPC-3 antibody for the active HCC targeting, a polymeric poly(lactic-co-glycolic acid) (PLGA)-based nanocarrier, and gold nanoparticles (GNPs) as imaging and therapeutic agents. Primarily, GPC-3 protein expression was assessed in different cancer cell lines (GPC-3 positive and negative), demonstrating the utility of GPC-3 as a suitable target and TAA for HCC. Hence, to obtain a specific custom-made anti-GPC-3 antibody, the production of mouse IgG monoclonal antibodies was implemented leading to the selection of D8 antibody being the most promising in terms of GPC-3 recognition and production yield. Following the characterization of D8 antibody, that revealed comparable performances as compared to clinical-used GC33 antibody, it was selected to be functionalized onto the NPs surface. PLGA nanocarrier was synthesised and in vitro characterized demonstrating excellent physiochemical properties. PLGA-NPs showed an average diameter of 250 nm, spherical nanocapsular morphology, thermal, pH and over time stability, absent cytotoxicity, and great ability to be internalized into HCC cells. As theranostic element, hydrophilic 14-15 nanometer-sized GNPs were chemically produced and characterized, and therefore encapsulated inside the PLGA-NPs core (hybrid NPs). The encapsulation process was confirmed by means of several techniques, and the gold amount quantified for in vivo applications. To test the contrasting ability, hybrid NPs were analyzed by X-ray Computed Tomography showing promising results in the potential to be undoubtedly distinguished from surroundings especially when resuspended in a membrane matrix mimicking the biological environment. Finally, the hybrid NPs were covalently functionalized with D8 antibody and in vivo and ex vivo investigated. When intravenously (IV) injected in HCC xenograft mouse model following the biodistribution for 48h timepoint, targeted and untargeted hybrid NPs mainly accumulated in elimination organs (liver and spleen) to a larger extent as compared to GNPs alone. However, D8 hybrid NPs exhibited a higher accumulation and retention in the tumor mass corroborating the benefit of an active cancer targeting approach. Despite the encouraging results obtained, the proposed system should be better characterized and investigated. Nevertheless, it lays the groundwork for potential in vivo application in clinical setting.
19-mag-2023
35
2021/2022
Settore BIO/14 - Farmacologia
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3048162
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