Hyaluronic Acid Vehicles for Cell Targeting

Abstract

This thesis discusses the use of hyaluronic acid (HA) drug delivery vehicles as potential targeted therapies for cancer and inflammatory disease. We focus on the molecular design and processing of HA materials driven by the overexpression of its main endocytic receptor, CD44, in most solid tumors and the inflamed milieu. In particular, we have an interest in hijacking the natural mechanism of CD44-mediated HA endocytosis to deliver active pharmaceutical ingredients; however, CD44 has been described as "a molecule with a thousand faces" and the design of targeted strategies requires a solid understanding of its intricate cellular processing. As such, a comprehensive review is covered in Chapter 1. Within the framework of CD44-targeted delivery, our group has previously developed a nucleic acid delivery platform based on HA-exposing chitosan nanoparticles prepared by ionotropic gelation. In Chapter 2 we explored a simplified preparative method yielding nanoparticles with virtually identical physico-chemical properties and delivery capabilities. This procedure considerably improves the manufacturing process in terms of timing and risks. Additionally, it avoids loss of the nucleic acid payload, guarantees sterility and improves reproducibility and scalability. Further evaluation of these particles was carried out in Chapter 3, where we looked at the complex relationship between THP-1 macrophage polarization and the uptake of HA materials. We demonstrated that, paradoxically, a higher CD44 expression (M1 - classical activation) allows a more efficient capture of HA carriers, but a lower expression (M2 - alternative activation) is conducive to better internalization. In Chapter 4 we moved on to evaluate the transfection efficiency effects of chitosan macromolecular parameters by producing a library of nanoparticles that differed both in RNA binding strength (avidity) and in their internalization rate in HCT-116. Interestingly, we showed that the increase in chitosan molecular weight was detrimental for RNA release, had a complex influence on internalization rate, but proved a very positive factor for transfection efficiency. We ascribe this to an improved RNA protection and enhanced endosomolytic activity. The targeting behavior of the best performing formulations was finally assessed in Chapter 5, which explores feasibility of using chitosan/HA nanoparticles to preferentially deliver a siRNA payload into cancer cells (over normal cells). We found that CD44 expression correlated with nanoparticle internalization and silencing efficiency, overall higher in cancer cell lines; however, we depicted a remarkable lack of correlation in HT-29 colorectal cancer cells despite their high CD44 expression. We believe the puzzling behavior of this cell line is a wake-up call for researchers in the field to stress the role of the activation state and internalization capabilities of CD44 and of its variant isoforms rather than predicting the success of targeted drug delivery based on the overexpression of HA receptors alone.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Stratford (Supervisor), Kaye Williams (Supervisor), Nicola Tirelli (Supervisor) & Annalisa Tirella (Supervisor)