Periodic Reporting for period 1 - HypoCyclo (Multifunctional Polymeric Cyclodextrin Nanocarriers As Novel Triple-Negative Breast Cancer Treatment: A Versatile Photo-Chemotherapy Bypassing Hypoxic Conditions)
Reporting period: 2020-09-01 to 2023-08-31
Among all breast cancers affecting female population worldwide, 20% is represented by Triple Negative Breast Cancer (TNBC), an aggressive subtype with very limited therapeutic options and poor survival rates. There is a continuous search for new therapeutic approaches that can overcome the shortcomings of the actually used therapies, mainly surgery and chemotherapy. Seeking for innovation in TNBC therapies, the HypoCyclo project focuses the optimal combination of chemotherapy and photodynamic therapy (PDT). PDT, a less known therapeutic approach, consists in the administration of a molecule absorbing red light, known as "photosensitizer" which produces reactive oxygen species (ROS), mainly singlet oxygen, upon irradiation with red light in the presence of oxygen. ROS can start a cascade of biological processes eventually killing cancer cells. PDT has become a feasible option thanks to the technological advances in lasers and fiber-optics allowing interstitial and intra-operative light delivery for the treatment of solid tumours, including breast cancers. The presence of oxygen is necessary for both chemotherapy and PDT to be effective. However, breast cancers often suffer from low oxygen levels, called hypoxia, limiting efficacy of the chosen therapies and therefore, and new systems that are able to release oxygen at the cancer site are currently investigated.
The objective of the project is the combination of chemotherapy with photodynamic therapy in optimized conditions for the effective treatment of TNBC also in hypoxic conditions. We plan to prepare new polymeric nanoparticles able to load cancer drugs together with a photosensitizer for PDT as well as an oxygen releasing agent (ORA). The goal of the project is also to keep the synthetic protocols simple, low-cost and green, in order to facilitate industrial application of the nanoparticles that perform best.
The objective of the project is the combination of chemotherapy with photodynamic therapy in optimized conditions for the effective treatment of TNBC also in hypoxic conditions. We plan to prepare new polymeric nanoparticles able to load cancer drugs together with a photosensitizer for PDT as well as an oxygen releasing agent (ORA). The goal of the project is also to keep the synthetic protocols simple, low-cost and green, in order to facilitate industrial application of the nanoparticles that perform best.
We prepared cyclodextrin (CyD)-based polymeric nanoparticles (NPs) by polycondensation of native cyclodextrins in the presence of a cross linking agent, epichlorohydrin. They were further modified with positively-charged quaternary ammonium groups. Cyclodextrins have been chosen as they are biocompatible and endowed with excellent features as host systems for water-insoluble drugs. We selected the beta-cyclodextrin polymers for the loading of three components: (1) the drug paclitaxel (PCX) used for TNBC treatment, presenting several treatment problems like water insolubility, (2) Chlorin e6, a photosensitizer currently investigated clinically for PDT, and the (3) molecular system able to release oxygen (ORA). As ORA we prepared photochemically 2 new endoperoxides starting from anthracenes while the same method resulted not to be successful with naphthalenes. Thanks to the complexation in the CyD polymer the photochemical preparation of the anthracenic endoperoxides was obtained also in water. Only one anthracenic endoperoxide is able to release oxygen upon heating and UV irradiation.
The CyD polymer NPs successfully loaded the drug PCX, the photosensitizer Chlorin e6 and the anthracenic endoperoxide and the loaded NPs were completely characterized. First cell studies indicated that the neutral beta-CyD polymer is well tolerated while the positively-charged CyD polymer is more cytotoxic and thus not further explored. Currently the efficacy of the loaded system is investigated in BC cell lines.
The CyD polymer NPs successfully loaded the drug PCX, the photosensitizer Chlorin e6 and the anthracenic endoperoxide and the loaded NPs were completely characterized. First cell studies indicated that the neutral beta-CyD polymer is well tolerated while the positively-charged CyD polymer is more cytotoxic and thus not further explored. Currently the efficacy of the loaded system is investigated in BC cell lines.
We significantly optimized straightforward synthesis of aromatic substrates to be used for the preparation of oxygen releasing agents (ORAs) compared to published procedures. The synthesized substrates, two pairs of di- and mono-substituted anthracene (PABA, ANBA) and naphthalene (PNBA, NABA) derivatives were subject of photooxygenation in the presence of methylene blue (MB) as low-cost PS in a mixture of organic solvents, reaching the goal of endoperoxidation only for the anthracenes.
Using the cyclodextrin-based polymer, the aromatic molecules were effectively dissolved in the mM range in water. After co-encapsulation of catalytic amounts of MB into the polymer and following the same protocol used in the case of organic solvents, the anthracenes ANBA and PABA were photochemically transformed in their endoperoxide analogues in PBS buffer (pH 7.4) of both D2O and H2O. This latter represents an extraordinary result as singlet oxygen has a much shorter lifetime in this solvent. Naphthalene-substrates, instead, were not suitable for photooxygenation neither in aqueous solvent. Cycloreversion of PABA-O2 was achieved upon heating in both organic and aqueous environments even though in conditions not yet compatible with in vivo applications.
Hence, the polymer acts both as solubilizing agent and reaction vessel enabling the photooxygenation in buffered water of hydrophobic substrates with an economical setup, comparable concentrations and improved reaction times with respect to the organic solvent. These results are of great importance for future exploitations in green synthesis and have the potential to launch CyD polymers as affordable hydrophilic platform for the development of more sustainable homogeneous photocatalytic protocols. Exploiting the versatility of the polymer to simultaneously solubilize different hydrophobic substrates bringing them in close proximity, also other types of reactions can be envisaged on hydrophobic molecules in aqueous environment. Moreover, once optimized the cycloreversion conditions, the use of a unique inexpensive polymeric matrix to solubilize, synthesize and deliver ORAs might definitively push the fast developments of new treatments for diseases displaying hypoxic environments.
Using the cyclodextrin-based polymer, the aromatic molecules were effectively dissolved in the mM range in water. After co-encapsulation of catalytic amounts of MB into the polymer and following the same protocol used in the case of organic solvents, the anthracenes ANBA and PABA were photochemically transformed in their endoperoxide analogues in PBS buffer (pH 7.4) of both D2O and H2O. This latter represents an extraordinary result as singlet oxygen has a much shorter lifetime in this solvent. Naphthalene-substrates, instead, were not suitable for photooxygenation neither in aqueous solvent. Cycloreversion of PABA-O2 was achieved upon heating in both organic and aqueous environments even though in conditions not yet compatible with in vivo applications.
Hence, the polymer acts both as solubilizing agent and reaction vessel enabling the photooxygenation in buffered water of hydrophobic substrates with an economical setup, comparable concentrations and improved reaction times with respect to the organic solvent. These results are of great importance for future exploitations in green synthesis and have the potential to launch CyD polymers as affordable hydrophilic platform for the development of more sustainable homogeneous photocatalytic protocols. Exploiting the versatility of the polymer to simultaneously solubilize different hydrophobic substrates bringing them in close proximity, also other types of reactions can be envisaged on hydrophobic molecules in aqueous environment. Moreover, once optimized the cycloreversion conditions, the use of a unique inexpensive polymeric matrix to solubilize, synthesize and deliver ORAs might definitively push the fast developments of new treatments for diseases displaying hypoxic environments.