Periodic Reporting for period 1 - POLAR STAR (Polymeric cyclodextrin-based nanoparticles for combination therapy of castrate-resistant prostate cancer)
Reporting period: 2019-06-01 to 2021-05-31
With about 375 000 deaths worldwide in 2020, PC is the fifth leading cause of cancer-related death in the male population. Localized PC can be successfully cured by radical surgery or radiation therapy while the advanced and metastatic PC diseases are treated mainly with androgen deprivation therapy (ADT). ADT-sensitive PC often evolves into Castration Resistant Prostate Cancer (CRPC). The lack of efficacious treatments for CRPC has led to an urgent need for the development of new therapies. These will likely require personalized therapy based on combination therapy with drugs targeting different metabolisms specifically operating in CRPC. Administration of multiple therapeutics can be difficult because of unfavorable distribution in the body, rapid body clearance, limited tumor accessibility and toxicity. In this frame, nanotechnology for drug delivery can offer a series of advantages such as the possibility to load in a unique nanoparticle system high amounts of drugs as well as the combination of more than one therapeutic agent. POLAR STAR aims at the implementation of combination therapy to treat CRPC. Our strategy is to exploit nanotechnology to administer contemporarily different therapeutic agents that synergically act across multiple oncogenic pathways. We plan to use new polymers based on cyclodextrins (CyD) as these are already FDA approved. The Polar Star Objective is the combination of known antitumoral drugs with “less known” therapeutic agents as it may open new roads for innovation in CRPC treatment. As to “less known” therapeutics, we are interested in agents able to produce cytotoxic species upon light irradiation. The idea is to combine known drugs together with a photosensitizer (PS) which upon irradiation with red light in the presence of oxygen produces Reactive Oxygen Species starting a cascade of processes finally killing the cancer cells. To implement combination therapy we aim at the preparation of nanoparticles (NPs) of polymerized CyDs able to encapsulate both the drugs and the PS. The loaded polymeric NPs will be further studied in biological assays with the goal to assess toxicity on normal and tumor prostate cells. We plan to obtain cell proliferation profiles for the various NP formulations. We also aim at the rationalization of the biological response of 3D PC organoids after treatment with the loaded NPs. Our final goal is the preparation of different NPs that encapsulates the right amounts of drugs and photosensitizer that are ready for the application in vivo.
The Polar Star project was launched in June 2019. Progress of the project has been severely compromised by the COVID-19 pandemics since its outbreak in Italy in February 2020. Access to the lab facilities near the CNR was strongly limited since then. Further, travel restrictions did not allow the IF to make two secondments near CycloLab in Budapest and near the Erasmus Medical Center (EMC) in Rotterdam. Consequently, only a part of the objectives was achieved as we will describe below.
The work was organized into four actions: i) synthesis of the polymeric carriers, ii) Loading of the carriers with multiple therapeutics, iii) Biological assays of the new drug loaded carriers, iv) Study of the drug/carrier systems in PC 3D models. In short, WP1 and WP2 activities have been completed satisfactorily, while the activities of WP3 were initiated in collaboration with the EMC and the University of Bologna, and those of WP4 were not executed due to time shortage. These last activities are currently ongoing near EMC in Rotterdam.
Results:
WP1 Synthesis of CyD polymers: The partner CycloLab Srl prepared new CyD polymers using a consolidated protocol based on polycondensation of natural CyDs in the presence of epichlorohydrin in aqueous alkaline environment. The synthesis has already been reported for polymers of single CyD and they used the same approach to prepare mixed CyD polymers.
WP2 Preparation and characterization of the new drug/carrier assemblies: We used 5 polymers, prepared in WP1, paCyD, pbCyD, pgCyD, pabCyD, and pbgCyD. We focused on drugs proposed for the treatment of CRPC like Cabazitaxel, Bicalutamide and Enzalutamide. These drugs display serious drawbacks related with their low aqueous solubility and with the onset of severe side effects, forcing administration in tolerable doses. As photosensitizer we considered Mitoxantrone, a cancer drug, and Chlorin e6, a well known singlet oxygen sensitizer. Electronic absorption spectroscopy was used to estimate the amount of loaded drug and circular dichroism and fluorescence were further exploited to confirm encapsulation. We obtained the maximum solubility data for cabazitaxel, bicalutamide and enzalutamide or their combinations. We collected the binding constants for the drug/carrier systems displaying the most interesting solubility profile. This WP was completed with a study of the interaction of the polymers with Mitoxantrone and chlorin e6. Only Chlorin e6 exhibits good affinity for the polymers. For the polymers displaying the best profile in solubilizing a drug we assessed the stability of the solution over time. We used DLS to investigate the dimension of the loaded polymers assembling in particles with a size below 20 nm.
WP3 Biological 2D assays: More than one drug/carrier combination emerged from WP2 and in the short time left we performed the first biological tests with the pbCyD solubilizing BIC, CBX and both in collaboration with UNIBO and the EMC where CRPC cell lines are available. The first results obtained with MTT assay on a CRPC cell line show that cell proliferation is similar for the drugs alone and for the drugs loaded by the polymer. Further investigations are required to complete the data with the other dug carrier systems as well as the investigation of the phototoxicity when one of the drugs is coloaded with chlorin e6.
WP4:Biological performance of the new drug/carrier systems in 3D PC models: This WP has not been addressed up to now.
The work was organized into four actions: i) synthesis of the polymeric carriers, ii) Loading of the carriers with multiple therapeutics, iii) Biological assays of the new drug loaded carriers, iv) Study of the drug/carrier systems in PC 3D models. In short, WP1 and WP2 activities have been completed satisfactorily, while the activities of WP3 were initiated in collaboration with the EMC and the University of Bologna, and those of WP4 were not executed due to time shortage. These last activities are currently ongoing near EMC in Rotterdam.
Results:
WP1 Synthesis of CyD polymers: The partner CycloLab Srl prepared new CyD polymers using a consolidated protocol based on polycondensation of natural CyDs in the presence of epichlorohydrin in aqueous alkaline environment. The synthesis has already been reported for polymers of single CyD and they used the same approach to prepare mixed CyD polymers.
WP2 Preparation and characterization of the new drug/carrier assemblies: We used 5 polymers, prepared in WP1, paCyD, pbCyD, pgCyD, pabCyD, and pbgCyD. We focused on drugs proposed for the treatment of CRPC like Cabazitaxel, Bicalutamide and Enzalutamide. These drugs display serious drawbacks related with their low aqueous solubility and with the onset of severe side effects, forcing administration in tolerable doses. As photosensitizer we considered Mitoxantrone, a cancer drug, and Chlorin e6, a well known singlet oxygen sensitizer. Electronic absorption spectroscopy was used to estimate the amount of loaded drug and circular dichroism and fluorescence were further exploited to confirm encapsulation. We obtained the maximum solubility data for cabazitaxel, bicalutamide and enzalutamide or their combinations. We collected the binding constants for the drug/carrier systems displaying the most interesting solubility profile. This WP was completed with a study of the interaction of the polymers with Mitoxantrone and chlorin e6. Only Chlorin e6 exhibits good affinity for the polymers. For the polymers displaying the best profile in solubilizing a drug we assessed the stability of the solution over time. We used DLS to investigate the dimension of the loaded polymers assembling in particles with a size below 20 nm.
WP3 Biological 2D assays: More than one drug/carrier combination emerged from WP2 and in the short time left we performed the first biological tests with the pbCyD solubilizing BIC, CBX and both in collaboration with UNIBO and the EMC where CRPC cell lines are available. The first results obtained with MTT assay on a CRPC cell line show that cell proliferation is similar for the drugs alone and for the drugs loaded by the polymer. Further investigations are required to complete the data with the other dug carrier systems as well as the investigation of the phototoxicity when one of the drugs is coloaded with chlorin e6.
WP4:Biological performance of the new drug/carrier systems in 3D PC models: This WP has not been addressed up to now.
Scientific Impact: Despite the above-mentioned problems, the work performed has led to the development of some novel carriers efficiently loading two drugs of clinical interest acting on different oncogenic pathways. They allow the contemporary administration of the drugs and resolve problems caused by the drug insolubility in physiological fluids.These results can find exploitable applications in CRPC treatments and represent a progress in the field chemotherapy for CRPC.
Impact on the career of the IF: The training scheme of the IF was outlined in the career development plan signed in September 2019 by IF and supervisor. The proposed training program has been partially fulfilled. The IF addressed new research topics, and consolidated her skills in pharmaceutical chemistry; gained experience in the use of optical spectroscopic equipment; received her first training in time-resolved confocal fluorescence imaging; actively participated in project management; was in charge of the experimental tasks and monitored achievement of milestones in close interaction with the supervisor; submitted a research proposal towards the end of the project to get funding from AIRC; participated in the preparation of a research proposal submitted by her supervisor; improved her communication skills targeting both scientific and wide public. She left one month earlier than foreseen thanks to a new job opportunity near a private company.
Impact on the career of the IF: The training scheme of the IF was outlined in the career development plan signed in September 2019 by IF and supervisor. The proposed training program has been partially fulfilled. The IF addressed new research topics, and consolidated her skills in pharmaceutical chemistry; gained experience in the use of optical spectroscopic equipment; received her first training in time-resolved confocal fluorescence imaging; actively participated in project management; was in charge of the experimental tasks and monitored achievement of milestones in close interaction with the supervisor; submitted a research proposal towards the end of the project to get funding from AIRC; participated in the preparation of a research proposal submitted by her supervisor; improved her communication skills targeting both scientific and wide public. She left one month earlier than foreseen thanks to a new job opportunity near a private company.