Reversion of P-gp-Mediated Drug Resistance in Ovarian Carcinoma Cells with PHPMA-Zosuquidar Conjugates

Inhibition of P-glycoprotein (P-gp) transporter is an attractive approach for the reversion of cancer-associated multidrug resistance (MDR). Poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA)-based carriers that are able to release the anticancer drug doxorubicin in the lysosomes have shown promise to reduce P-gp mediated resistance. This is attributed to the release of the drug in close proximity to the nucleus and distant from the P-gp transporter. This work presents a strategy to maximize P-gp inhibition and enhance doxorubicin cytotoxicity in cancer cell by using a dual functional PHPMA conjugate carrying both the anticancer drug doxorubicin and the P-glycoprotein inhibitor zosuquidar (Zos). While doxorubicin was connected to the polymer backbone via a lysosomally cleavable spacer, the P-gp inhibitor Zos was attached by a hydrazone linker in order to promote release in the early stage of the endocytic process and maximize its,cytosolic, concentration in proximity of P-gp transporter. Following Zos modification and determination of its ability to inhibit P-gp, conjugation to the PHPMA polymer backbone resulted in enhanced doxorubicin cytotoxicity in resistant A2780ADR ovarian carcinoma cells. Finally, the incorporation of both Dox and Zos in a single polymer carrier enhanced P-gp inhibition as compared to a control PHPMA conjugate containing only Dox.

Controlling and Monitoring Intracellular Drug Delivery Using PHPMA-Based Polymer Nanomedicines

Claudia Battistella

Polymer nanomedicine is an attractive approach for the delivery of anticancer drugs. Firstly designed to increase drug bioavailability, polymer conjugates and polymer nanoparticles have rapidly emerged in the field of cancer therapy after the discovery of the enhanced permeation and retention (EPR) effect. The leaky and disordered tumor neovascularization provides opportunities to guide the accumulation of polymer nanomedicines to the tumor tissue therefore enhancing therapy selectivity and reducing off-target-associated side effects. However, since many chemotherapeutics act on targets that are located in well-defined subcellular compartments, controlling the intracellular fate of polymer nanomedicines and/or their payload is another important factor that contributes to therapy efficiency. Polymer conjugates and polymer nanoparticles generally access the cell interior via endocytosis. The physiochemical and biochemical parameters that distinguish the endolysosomal compartments from the extracellular environment have been widely exploited to trigger intracellular drug release from the polymer carriers. Amongst a range of other polymers and polymer nanoparticles that have been investigated over the past 30 years, poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA)-based conjugates have been extensively explored for the endolysosomal release of anticancer drugs. While the modern polymer chemistry toolbox provides many opportunities to tailor the molecular weight and functionality of PHPMA and to introduce features that allow the polymer to respond to the different endolysosomal environments, the development of tools and methods to monitor these processes is also crucial for the future development of advanced delivery systems. The aim of this Thesis is to design dual-functional PHPMA polymers that offer the possibility to control the endolysosomal release of anticancer drug combinations as well as to monitor the PHPMA endolysosomal trafficking. Chapter 1 of this Thesis provides an overview of the different approaches that have been described in literature to control and monitor the intracellular delivery of polymer nanomedicines. Chapter 2 describes a synthetic approach to prepare alpha- and alpha,omega-fluorine labeled pentafluorophenyl methacrylate (PPFMA) polymers via reversible addition fragmentation chain transfer (RAFT) polymerization. In Chapter 3 post-polymerization modification of the alpha-fluorine labeled PPFMA precursor will be used to prepare a series of PHPMA conjugates carrying either the anticancer drug doxorubicin (Dox) or the P-glycoprotein inhibitor zosuquidar (Zos) or both drugs at the polymer side chains. The ability of the conjugates to overcome doxorubicin efflux and therefore reverse P-gp-mediated multidrug resistance in resistant ovarian carcinoma cells will be assessed. Finally, Chapter 4 will study the cellular internalization and endolysosomal trafficking of PHPMA. The synthesis of a dual-labeled PHPMA polymer containing both a fluorescent as well as a fluorinated label will be used for flow cytometry and confocal fluorescence microscopy studies and to demonstrate the potential of nanoscale secondary ion mass spectrometry (NanoSIMS) to map and localize fluorine-containing polymers in cells.

EPFL2017

Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors

Anne Planche, Tania Santoro

Background: Blocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study. Methods: Cancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis. Results: Exposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment. Conclusions: Taken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

Biomed Central Ltd2016

Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors

Anne Planche, Tania Santoro

Biomed Central Ltd2016

Controlling and Monitoring Intracellular Drug Delivery Using PHPMA-Based Polymer Nanomedicines

Claudia Battistella

EPFL2017