(291d) Dual Responsive Backbone Shattering Organelle Targeted Nano-Prodrug to Modulate Oxygen Consumption for Effective Chemophototherapy

The clinical translation of nanomedicine for glioblastoma multiforme (GBM) faces four key challenges: (1) lack of precise control over carrier architectures, drug composition and batch-to-batch variations in drug loading content/ release profile, (2) lack of a technique to monitor the fate of the payload without using any substitute labelling molecule, (3) over dependence on monotherapy to treat heterogeneous solid tumours thereby contributing to therapeutic resistance, (4) suboptimal therapeutic concentrations in the brain tumour due to the presence of Blood Brain Barrier (BBB). Photodynamic therapy is a promising clinical alternative to conventional treatments such as chemotherapy and radiotherapy. However, its dependence on oxygen limits its therapeutic potential in hypoxic tumours. To address this issue, several strategies have been studied including direct oxygenation of the tumour using components such as enzymes and perfluorocarbon, usage of red blood cells and haemoglobin as oxygen carriers. However, the realization of a satisfactory photodynamic therapeutic effect remains challenging due to irregularity in tumour blood vessels which leads to heterogeneous oxygen supply and uneven photosensitizer distribution. Recently, modulation of oxygen consumption has received considerable attention as an approach that reduces oxygen partial pressure gradients within tumours, thereby alleviating hypoxia in poorly vascularized regions. In this study, we report the design and synthesis of a dual responsive nano-prodrug (DRnp) that not only efficiently generates reactive oxygen species (ROS), but also specifically releases its chemotherapeutic module on demand in the tumour microenvironment. DRnp is self-assembled from an amphiphilic polymer brush, which comprises of a redox responsive photodynamic backbone grafted with poly(ethylene glycol) and conjugated with a pro-apoptotic peptide through a light responsive bond. DRnp is used to encapsulate an oxygen regulator, atovaquone. Furthermore, the cell penetrating peptide CGKRK targets the angiogenic blood tumour blood vessels to significantly improve the vascular damage through photodynamic therapy, while the proapoptotic D-amino acid peptide KLAKLAKKLAKLAK disrupts the mitochondrial membrane thereby promoting apoptotic cell death. A photosensitizer Protoporphyrin IX (PpIX) was polymerized with a redox responsive linker cystamine dihydrochloride which was further conjugated to PEG and a cell penetrating pro-apoptotic peptide (CGKRK_D(KLAKLAK)₂) using a ROS responsive thioketal linker. The synthesized material was characterized using NMR and GPC. DRnp was obtained by a nanoprecipitation method. The dual responsive ability of DRnp were studied in different pH and GSH environments. The ability of DRnp to pass the BBB was studied using an in-vitro BBB model. Finally, the penetration and cytotoxicity of DRnp were studied in an U87 and 3T3 co-culture tumour spheroid model. GPC data showed that the number average molecular weight of DRnp was circa 120,000 with good polydispersity (PDI). TEM showed DRnp has a uniform spherical morphology with an average diameter of ~120 nm, consistent with the results from dynamic light scattering. Significant penetration and cytotoxicity of DRnp was observed in multicellular spheroids after passing through the in-vitro BBB. The distinctive features of DRnp include (1) photosensitizer and chemotherapeutic peptide were combined at a fixed ratio for precise nanomedicine, (2) the photodynamic backbone was site specifically shattered at high GSH concentrations along with ROS mediated cleavage of the therapeutic peptide in the acidic tumour microenvironment, (3) Can pass through the BBB, and (4) minimizes the oxygen consumption rate within solid tumours. Overall, DRnp enables site specific integrated PDT and chemotherapy for synergistically amplified cancer treatment.