Multifunctional Magnetic Nanoparticles for Hyperthermia based Hypoxic Tumor Therapy

Abstract

Hypoxia, a state of reduced oxygen availability or decreased oxygen partial pressure is a common characteristic in many types of solid tumors. Hypoxic tumor microenvironment (TME) significantly contributes to the resistance of tumor cells against various therapeutic modalities that rely on molecular oxygen for their effectiveness such as radiotherapy, chemotherapy, immunotherapy and photodynamic therapy. Recent advancements in nanotechnology have provided novel perspectives, however, current nanocarriers demonstrate limited effectiveness in reaching hypoxic regions due to their dependency on systemic circulation, lack of a propulsive force to penetrate beyond diffusion limits, and the absence of sensory based displacement capabilities to target the hypoxic zones. Consequently, it is important to find alternative strategies to mitigate tumor hypoxia using advanced nanotherapeutics capable of overcoming the present limitations in hypoxic cancer nanotherapy. On the other hand, magnetic hyperthermia-mediated cancer therapy (MHCT), is a therapeutic approach that involves the localization of magnetic nanoparticles (MNPs) within the tumor site. On the application of an alternating magnetic field (AMF), the oscillation of magnetic moments in MNPs generates a localized temperature rise, selectively killing heat-sensitive tumor cells with minimal adverse effects on healthy cells. This heat induces tumor cell death by activating certain intracellular and extracellular degradation mechanisms. However, the ineffective penetration of MNPs into hypoxic tumor cores due to pathological pressure gradients, such as excessively high interstitial fluid pressure and solid tissue pressure, poses a significant obstacle to MHCT. Therefore, to enhance the therapeutic efficacy of nanocarriers in hypoxic tumor microenvironments, it is essential to develop agents specifically targeting these regions that possess self-propelling capabilities. The present thesis focuses on the development and application of hypoxia-responsive magnetic nanomaterials as efficient magnetic hyperthermia agents for the successful inhibition of hypoxic solid tumors. With the primary objective to improve both the targeted delivery of these MNPs to solid tumors and to increase their therapeutic efficacy, we focused on the development of iron oxide nanoparticles with hypoxia specific penetration for heat-based therapy. To achieve this, E. coli was employed as a carrier for the delivery of MNPs to hypoxic tumor regions, owing to its tumor-colonizing ability. A detailed investigation was carried out by engineering E. coli to fabricate with an aim to evaluate the MHCT potential of these hybrid bacteria based magnetic nanoparticle system (BacMags) as effective delivery and therapeutic agents for cancer therapy. The use of tumor vii homing properties of E. coli enabled deep penetration into tumors, while anisotropic shaped iron-oxide nanocubes offered high heating efficiency. The efficacy of the bacteria-based approach to deliver MNPs in hypoxic tumors was further validated in a C57/BL6 mice model, demonstrating a complete tumor regression within one month of hyperthermia treatment. To further augment the heat-based killing effect generated by the MNPs, the surface of the MNPs was modified with polydopamine (PDA) coating to enhance their heat-based therapeutic applications. The incorporation of a PDA shell on the magnetic nanocubes imparts them the ability to respond to near-infrared (NIR) laser irradiation for photothermal ablation therapy (PTT) of tumors. Therefore, the PDA-coated MNPs bio-conjugated on E. coli served as effective thermo-therapy agents, demonstrating a synergistic magnetic hyperthermia and photothermal therapy (PTT) response. The dual-heat modality improved the cancer therapy outcome with a complete tumor regression in c57BL/6 mice within 20 days of treatment and a tumor free survival for 60 days without recurrence. We also focused on the design of an alternative approach to enhance the therapeutic efficacy of MNPs using the in-situ oxygen generating ability of two - dimensional (2D) Ni-Fe-layered double hydroxide (LDH) nanoflakes. A nanocomposite comprising of a photo-sensitiser agent (chlorin e6) intercalated with the Ni-Fe-LDH nanosheets were attached with the iron oxide nanocubes that were further loaded with a hypoxia-activated pro-drug, tirapizamine (TPZ) for a multi-modal anti-cancer therapy. The ROS generation ability of the developed layered double-hydroxide /magnetic nanocubes/ chlorin e6 /tirapazamine (LNCT) nanocomposite along with its cellular uptake, heat triggered drug release, cytoskeleton damage, morphological alterations in pancreatic cancer cells upon heat treatment under AMF and NIR laser irradiation were evaluated. The coupling of LDH with chlorin e6 provided the catalytic playground for ROS generation during the combined magnetic hyperthermia and photodynamic therapy (PDT). The hypoxic environment additionally activates the hypoxia-activated pro-drug, TPZ, leading to a highly efficient ROS-mediated apoptosis in pancreatic cancer cells. The gene expression profile of the major genes involved in the hypoxia-inducible factor (HIF) pathway and apoptotic cancer cell death pathway such as HIF-1 alpha, caspases, etc., were further investigated to understand the molecular mechanism responsible for the hypoxic tumor cell death