The Effect of Hyperthermia on Doxorubicin Therapy and Nanoparticle Penetration in Multicellular Ovarian Cancer Spheroids

Abstract

The efficient treatment of cancer with chemotherapy is challenged by the limited penetration of drugs into the tumor. Nanoparticles (10 – 100 nanometers) have emerged as a logical choice to specifically deliver chemotherapeutics to tumors, however, their transport into the tumor is also impeded owing to their bigger size compared to free drug moieties. Currently, monolayer cell cultures, as models for drug testing, cannot recapitulate the structural and functional complexity of in-vivo tumors. Furthermore, strategies to improve drug distribution in tumor tissues are also required. In this study, we hypothesized that hyperthermia (43°C) will improve the distribution of silica nanoparticles in three-dimensional multicellular tumor spheroids. Tumor spheroids mimic the functional and histomorphological complexity of in-vivo avascular tumors and are therefore valuable tools to study drug distribution. Ovarian cancer (Skov3) and uterine sarcoma (MES-SA/Dx5) spheroids were generated using the liquid overlay method. The growth ratio and cytotoxicity assays showed that the application of adjuvant hyperthermia with Doxorubicin (DOX) did not yield higher cell killing compared to DOX therapy alone. These results illustrated the role of spheroids in resistance to heat and DOX. In order to study the cellular uptake kinetics of nanoparticles under hyperthermia conditions, the experimental measurements of silica nanoparticle uptake by cells were fitted using a novel inverse estimation method based on Bayesian estimation. This was coupled with advection reaction transport to model nanoparticle transport in spheroids. The model predicted an increase in Area Under the Curve (AUC) and penetration distance (W1/2) that were validated with in-vitro experiments in spheroids. Based on these observations, a novel multifunctional theranostic nanoparticle probe was created for generating highly localized hyperthermia by encapsulating a Near Infrared (NIR) dye, IR820 (for imaging and hyperthermia) and DOX in Organically modified silica nanoparticles (Ormosil). Pegylated Ormosil nanoparticles had an average diameter of 58.2±3.1 nm, zeta potential of -6.9 ± 0.1 mV and high colloidal stability in physiological buffers. Exposure of the IR820 within the nanoparticles to NIR laser led to the generation of hyperthermia as well as release of DOX which translated to higher cell killing in Skov3 cells, deeper penetration of DOX into spheroids and complete destruction of the spheroids. In-vivo bio-distribution studies showed higher fluorescence from organs and increased plasma elimination life of IR820 compared to free IR820. However, possible aggregation of particles on laser exposure and accumulation in lungs still remain a concern.