Redox-Sensitive Liposomes - How Design of Redox-Sensitive Liposomes Affects in Vitro and in Vivo Behavior and Development of a Method to Study Intracellular Activation

Redox-sensitive nanoparticles is a fairly new and exciting drug delivery strategy that relies on extracellular or intracellular redox molecules for activation leading to increased uptake, endosomal escape, or drug release. Due to the involvement of the redox system in the etiology of several diseases, the approach has many applications although it has mainly been studied for anticancer treatment. Redox-sensitivity is mainly achieved through inclusion of disulfides in the construct that should be activateable. Although redox-sensitive nanoparticles have been studied intensively, there is still a lack of knowledge on how design of the redox-sensitive moiety and carrier formulation impacts important clinical parameters. In addition the cellular location of disulfide cleavage is debated, which means that the mechanistic behavior of redox-sensitive drug delivery systems is largely unknown.
In this dissertation, redox-sensitive liposomes were established through the insertion of redoxsensitive lipopeptides (RSLs) in the membrane to shield a positive charge that upon reduction of RSLs should lead to endosomal escape. RSL liposomes were characterized in vitro and in vivo to gain an understanding of characteristics that may impact the behavior and clinical applicability. In addition a fluorescent construct was synthesized to evaluate intracellular reduction. In the first study, RSL design along with liposomal formulation was observed to impact characteristics such as cellular uptake and charge-reversal and an optimal formulation was chosen for further experiments. In the second study, RSL liposomes were functionalized with an antibody and loaded with an anti-cancer drug. In vitro studies showed desirable stability characteristics comparable to a clinical relevant formulation (Stealth). Pharmacokinetics revealed that the redox-sensitive liposomes circulated to a lesser degree than Stealth, which lead to a lower degree of tumor control. However, targeted RSL liposomes showed superior tumor control compared to nontargeted RSL liposomes and targeted liposomes incorporating non-cleavable lipopeptides. Improved anti-cancer efficacy was thus observed to be due to a combined effect of targeting and redox-sensitivity. In the third study, intracellular reduction of the RSL liposomes was visualized through an activateable fluorophore. The intracellular activation of the fluorophore was a slow process and activation in solution was shown to be influenced by pH. The slow intracellular activation and the distinct appearance of the fluorescence as punctas indicated that reduction of the RSLs occurred in vesicles along the endosomal-lysosomal pathway. In conclusion, the results obtained in this dissertation show that several design parameters of redox-sensitive liposomes may affect the clinical effect and further characterization should be performed to ensure the successful translation from preclinical experiments to the clinic.