(226b) Convection Enhanced Delivery of Light Responsive Antigen Capturing Oxygen Generators for Photoimmunotherapy of Hypoxic Tumors

The combination of photo- and immunotherapy presents a promising strategy to overcome the existing challenges with current clinically practised treatments. However, the reliance on oxygen proves to be the primary determinant of the therapeutic effect against tumour hypoxia. The resulting oxygen consumption and vascular damage due to photodynamic therapy (PDT) further exacerbates the tumour hypoxia and decreases its anti-tumour efficacy. Furthermore, the violent anti-tumour immune response elicited by the tumour-associated antigens due to PDT induced immunogenic cell death (ICD) is minimized due to the hypoxic tumour microenvironment. Hence, developing a stimuli responsive delivery system that enables on-demand drug release, modulates the tumour microenvironment by ameliorating hypoxia through in-situ oxygen generation and captures the tumour antigens can significantly magnify the therapeutic efficacy of photo-immunotherapy. In this work, we have developed a light responsive antigen capturing oxygen generator (LAG) co-loaded with a non-genotoxic molecule Nutlin-3a (NUT3a) and a potent photosensitizer Protoporphyrin IX (PpIX) to induce apoptotic cell death and create a specific anti-tumour immune response. An in-situ oxygen generating enzyme, catalase, was conjugated to polyethylene glycol and attached to 1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) through a reactive oxygen species (ROS) cleavable thioketal linker. Low energy light facilitated the on-demand release of NUT3a by leveraging on light induced reactive oxygen species (ROS) generation by PpIX and subsequent thioketal cleavage. NUT3a which promotes anti-tumour immunity and exerts precise toxicity towards wild-type p53 cancer cells while avoiding toxicity in immune cells and PpIX induces tumour regression through apoptosis, vascular damage and immune response. Furthermore, we have used multiphysics modelling to investigate convection enhanced delivery as an effective alternative to traditional delivery methods to overcome the blood brain barrier (BBB). The synthesis of LAG was confirmed using NMR. The dual drug loaded micelles were fabricated using a thin-film hydration method and the encapsulation efficiency and drug loading content was quantified using UV-Vis spectroscopy. The conjugation efficiency of catalase was evaluated using the Bradford assay. The cytotoxicity and penetration efficiency of the micelles were studied on 3D multicellular spheroids. The extent of immunogenic cell death was evaluated by measuring the expression of high-mobility group box 1 (HMGB1) and calreticulin (CRT). The antigen capturing capability of LAGs were assessed by monitoring the hydrodynamic diameter and zeta potential of LAGs along with the quantification of the proteins bound using the Bradford assay. The ability of the micelles to induce dendritic cell maturation was evaluated by flow cytometry. Furthermore, we studied the influence of treated tumour spheroids on DC maturation. The resulting T-cell proliferation was also investigated using flow cytometry. The cytokine production after incubation with Mo-DCs and spheroids were quantified by LEGENDplex assay. TEM images showed uniform spherical structures with a diameter of ~100 nm, similar to the results obtained using DLS. The encapsulation efficiencies of NUT3a and PpIX are ~78% and ~71% respectively. On irradiation, the dual drug loaded micelles showed superior cytotoxicity on U87 spheroids. The LAGs exhibited enhanced penetration and desirable distribution in the MCs compared to the non-targeted micelles and the free payloads. The targeted micelles showed an increase in the C83 and CD86 expressions compared to the other treatment groups, which was further enhanced in the presence of treated U87 spheroids. Overall, the developed system has the potential to effectively penetrate solid hypoxic tumours and enable precise delivery of the therapeutic molecules. The system leverages on the ability of NUT3a to induce apoptosis, cell cycle arrest and anti-tumour immunity through p53 activation, and PpIX to promote apoptosis through ROS generation, vascular damage and immune activation. This synergistic apoptosis and stimulation response has enormous potential for treatment and prevention of tumour reoccurrences.