PhD Thesis Defence Presentations - Dimitra Toumpa

Pancreatic cancer is one of the leading causes of cancer-related deaths in the Western world due to the lack of potent therapeutics and biomarkers for early diagnosis. A primary reason is the asymptomatic nature of the disease, as patients with stage III locally advanced pancreatic cancer (LAPC) exhibit severe symptoms only later, when the disease intersects with the celiac trunk or the superior mesenteric artery. Additionally, gemcitabine (GEM), one of the most commonly used monotherapies, is only marginally effective.

In recent years, combination therapies have improved survival rates in patients with various cancer malignancies. These therapies leverage the synergistic effects of multiple drug molecules targeting different pathways. Two combination therapies currently employed in clinical practice for LAPC are FOLFIRINOX (co-administration of folinic acid, fluorouracil, irinotecan, and oxaliplatin) and the co-administration of GEM with nab-paclitaxel (albumin-bound paclitaxel, nab-PTX). Despite the noticeable clinical benefit of the FOLFIRINOX regimen in improving overall survival, only a small number of patients can benefit from this therapy due to its high risk of lethal toxicity. To address these limitations, alternative strategies such as stimulus-responsive drug delivery systems are being explored to enhance efficacy while reducing systemic toxicity. Among these, ultrasound-mediated therapies have gained attention due to their non-invasive nature and ability to spatially control drug release. Ultrasound, a mechanical sound wave, can efficiently induce hyperthermia and generate cytotoxic reactive oxygen species (ROS). The combination of ultrasound waves with sonosensitizers (i.e., porphyrin molecules) to produce ROS is known as sonodynamic therapy (SDT). Although the mechanistic mode of action of SDT is not yet fully understood, it has shown promise in oncology for delivering small anticancer drugs and biologics (i.e., proteins, nucleic acids). Polymer-drug conjugates (PDCs) have recently garnered attention from the pharmaceutical industry, as they enable controlled and targeted drug release through the use of suitable linkers. Therefore, the aim of my thesis is to synthesize and biologically evaluate multidrug PDCs that release anticancer drugs via ultrasound stimulation.

Initially, commercially available monomer synthons were functionalized to attach drug molecules through covalent coupling. The choice of linker was a key consideration, as it plays a critical role in drug release. Accordingly, two types of linkers were synthesized: one containing disulfide bonds that can be easily hydrolyzed in cells via enzymes and acidic pH (the fast-reducible linker), and another with no sensitivity to changes in the cellular environment (the slow-reducible linker). Two anticancer drugs, GEM and camptothecin (CPT), were used, resulting in four hydrophobic monomer prodrugs that were ready for polymerization with various drug and linker combinations.

To investigate the release rates of the synthesized linkers and the efficacy of the drugs, these hydrophobic monomers were polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization with hydrophilic polyethylene glycol (PEG). Amphiphilic polymers were formed, and nanoparticles were generated through precipitation. Release studies demonstrated that drug release was enhanced under cancer cell culture conditions when the drug was attached to the disulfide-containing linker. Upon ultrasound irradiation, accelerated drug release was observed in these PDCs, which was further enhanced by the presence of a sonosensitizer due to ROS generation. Systematic in vitro testing across different treatment modalities revealed that some formulations were capable of outperforming the IC₅₀ values of the parent drugs by up to five orders of magnitude.

The drug-monomers were further combined with thermoresponsive polymers in a hyperthermia setting. Mild hyperthermia at 41–42°C has been shown to induce multifaceted effects on tumor physiology at both the tissue and cellular levels, including increased cell membrane fluidity, fragmentation of the endoplasmic reticulum, reduced ATP production, heat-shock protein overexpression, and activation of apoptotic caspase signaling pathways. Thus, thermoresponsive PDCs were developed to respond to mild hyperthermia conditions in combination with ultrasound activation. Starting from the polymerizable monomer-drug precursors, RAFT polymerization was performed with oligo(ethylene glycol) methyl ether methacrylate (OEGMA₅₀₀) and di(ethylene glycol) methyl ether methacrylate (MeO₂MA)—which have a lower critical solution temperature (LCST) of approximately 41–42°C, aligned with the desired hyperthermia range—yielding a library of thermoresponsive PDCs. Their release rates were evaluated above and below the LCST, with and without ultrasound, and in acidic or neutral pH. In vitro studies revealed that synergism was observed among the treatment modalities—PDCs, ultrasound, and temperature—with IC₅₀ values significantly lower than those of the parent drugs.

Finally, polyelectrolyte complexes (PECs) were developed to address critical challenges in drug delivery systems, such as improved biocompatibility and enhanced cellular uptake due to better interactions with cell membranes. Polyionic PDCs were synthesized using free radical polymerization (FRP), incorporating 2-(dimethylamino)ethyl methacrylate (DMAEMA) and HPMA-Suc to achieve positive and negative charges, respectively. These polyions were used to form PECs via nanoprecipitation through electrostatic interactions, and release experiments were conducted at two different pH levels. In vitro experiments demonstrated that enhanced cytotoxicity was achieved for PECs, particularly those containing CPT, along with low hemolysis, indicating their potential as novel nanomedicines that maximize therapeutic efficacy while ensuring safety.