STRENGTHEN OF DPNS FEATURES FOR THERANOSTIC APPLICATIONS AND MECHANICAL-CONTROL OF CHEMOTHERAPEUTIC EFFICACY THROUGH MODULATION OF CELL PROLIFERATION.

Solid tumors are complex biological structures which are composed of cellular and matrix components, everything being perfused by blood vessels. During tumor development, modifications of both biochemical and mechanical parameters are observed and can feedback on one another. Cancer cells constantly interact with their mechanical environment and the whole tissue is mostly confined by its surrounding. Compressive mechanical stress develops in part from cell proliferation and could eventually result in the clamping of blood vessels leading to increased interstitial fluid pressure (hydrostatic pressure). The consequent hypoperfusion poses important obstacles to drug delivery and nanomedicines. In fact, the tortuous tumor microvasculature has blood velocities up to one order of magnitude lower compared to healthy capillary networks. Moreover, the fast angiogenesis during tumor progression leads to high vascular density in solid tumors, large gaps exist between endothelial cells in tumor blood vessels, and tumor tissues show selective extravasation and retention of macromolecular drugs (Enhanced Permeation Retention – EPR – effect). These effects have served as a basis for the development of drug delivery systems which are aimed at enhancing tumor tissue targeting and drug therapeutic effectiveness. Over the last 15 years, a plethora of materials and different formulations have been proposed for the realization of nanomedicines. Yet, drug-loading efficiency, sequestration by phagocytic cells, and tumor accumulation of nanoparticle-loaded agents - nanomedicines - are sub-optimal. Starting from these considerations, during my PhD, I studied two complementary approaches: in the first two years my work was focused on implementing the characteristics of Discoidal Polymeric Nanoconstructs designed with controlled geometries and mechanical properties. In the last year, I investigated the role of mechanical stress on chemotherapeutic efficacy. More precisely, this work first reviews the use of deformable discoidal nanoconstructs (DPNs) as a novel delivery strategy for therapeutic and imaging agents. Inspired by blood cell behavior, these nanoconstructs are designed to efficiently navigate the circulatory system, minimize sequestration by phagocytic cells, and recognize the tortuous angiogenic microvasculature of neoplastic masses. In this work, the synthesis, drug loading and release, and physico-chemical characterization of DPNs were enhanced with particular emphasis on the ability to independently control size, shape, surface properties, and mechanical stiffness. Two different loading strategies were tested, namely the straightforward “direct loading” and the “absorbance loading”. In the former case, the agent was directly mixed with the polymeric paste to realize DPNs whereas, in the latter case, DPNs were first lyophilized and then rehydrated upon exposure to a concentrated aqueous solution of the agent. Under these two loading conditions, the encapsulation efficiencies and release profiles of three different molecules and their corresponding prodrugs were systematically assessed (1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine lipid chains or 1 kDa PEG chains were directly conjugated with Cy5.5 or methotrexate and Doxorubicin). Moderately hydrophobic compounds with low molecular weight showed encapsulation efficiencies of 80%, with absorption loading (direct loading has efficiencies around 1%). The DOX-DPN showed on triple negative breast cancer cells a toxicity comparable to free DOX. Preliminary in vivo preliminary studies conducted with directly loaded Cy5-DPN demonstrated a fairly solid integration of the imaging compound with the polymer matrix of the particles. The second part of the work dissect what happens to free drugs or to drugs carried by nanovectors once they reach the tumor site. As we mention above, the elevated mechanical stress derived from tumor progression could result in blood vessels clamping with consequent reduction of drug efficacy. It is quite obvious to imagine that if the drug fails to reach the tumor it cannot act on it. Indeed, mechanical stress within the tumor site is present from the early stages of the disease. Our goal was to understand what happens when mechanical stress is not yet so large enough to fully collapse the blood vessels. Are there mechanical alterations that can affect the efficacy of a chemotherapeutic? We studied how mechanical perturbations of the tumor microenvironment could contribute to the mechanical-form of Gemcitabine drug resistance. Specifically, we developed a new in vitro strategy to mimic the mechanical compression stress induced by the stroma during tumor progression. We embedded pancreatic tumor spheroids into agarose polymeric matrix in order to demonstrate the effect of mechanical compressive stress on tumor proliferation. Then, we validated our results with other types of mechanical stresses. Finally, we investigated the therapeutic efficacy of a proliferation-based chemotherapy: Gemcitabine. Collectively, having the physical cues of cancer in mind, it can be important to cross-fertilize the fields of physical oncology and nanomedicine.