Novel Multifunctional Cationic Lipid Hybrids for Therapeutic Applications in Cancer - Synthesis, Formulation, Characterization, and in vitro Drug Delivery

<p dir="ltr">Permanently charged cationic lipids are important excipients used in lipid-based drug and gene delivery systems. However, they can be toxic and are known to trigger unwanted biological responses including apoptosis, hemolysis, and immune responses via interactions with a variety of extracellular and intracellular proteins. They are also known to be rapidly cleared in vivo. Their toxicity is mainly attributed to their positively charged head group, although other molecular domains may also contribute. One strategy to harness the inherent toxicity of cationic lipids is via the rational design of lipids with cytotoxicity selectively targeted towards cancer cell lines. The main aim of this thesis is to design and synthesize permanently charged cationic lipids which are selectively cytotoxic by systematically altering the molecular architecture of the cationic lipid. Cubosome lipid nanocarriers are investigated throughout the thesis as potential drug delivery vectors for the synthesised cytotoxic cationic lipids, including in combination therapy with either a small molecule therapeutic or siRNA. As individual drugs differ in their pharmacokinetic properties due to differences in their solubilities it is ideal to combine them in a single nano vector at an appropriate ratio to achieve the desired therapeutic effect. Lipid based drug delivery systems are widely used for drug delivery purposes due to their biocompatibility. Cubosomes, which have a porous internal nanostructure of 3D cubic symmetry, are gaining increased attention in nanomedicine owing to a range of advantages. Their increased bilayer surface area allows higher loading of hydrophobic drugs. They have good thermodynamic stability, and their unique fusion mechanism of interaction with biological membranes may improve delivery efficacy of the encapsulated cargo. Research interest in cubosomes for the delivery of therapeutics and as vaccines has grown exponentially over the last two decades. Doping of cubosomes with cationic lipids (either permanently charged or ionizable) is increasingly used in drug and gene delivery as positively charged particles display improved cellular uptake and enhanced loading for anionic moieties such as RNA-based therapeutics and vaccines. </p><p dir="ltr">The thesis aims to answer the following research questions: 1. How does the structure of cationic lipids affect their cytotoxicity towards cancerous and non-cancerous cells? 2. Can the cytotoxicity of cationic lipids be improved via delivery in a cubosome nanocarrier? 3. How does the lipid structure affect the phase behaviour and cellular uptake of the cubosomes? 4. Can cubosomes functionalised with cationic lipids be used in combination therapy for the treatment of cancer cells? </p><p dir="ltr">Much of the research presented herein is based on an earlier research study from my research group (CSIR-IICT, Hyderabad, India) based on cationic lipid hybrids of anisic acid and cordiarimide. Lipid analogues were synthesised with a range of chain lengths from C2 to C18 and their cytotoxicity evaluated. Both the lipo-anisamide and cordiarimide analogues revealed moderate to potent anticancer activity in analogues with chain lengths in the range C7 – C14. Although C12M (lipo-anisamide) exhibited weak anticancer activity, NP C12 (lipo-cordiarimide) was observed to exert good anti-cancer activity against the liver cancer cell line Hep G2 and prostate cancer cell line PC-3. In addition, those analogues having a C12 carbon chain exhibited selective and efficient inhibition of human DNA ligase I (hLigI), an enzyme involved in DNA damage repair which is known to play a role in the development of cancer drug resistance. In contrast, little or no inhibition of other human ligases (hLigIIIβ and hLigIV/XRCC4) and bacterial T4 DNA ligase was observed. Thus, the 12 carbon chain length analogue, which demonstrated both reasonable anticancer activity and selective inhibition hLig I, was selected as optimal for the synthesis of the cationic lipids tested in this thesis. Herein, the data obtained in this previous research study was extended towards the synthesis of four novel cationic lipids with potential anticancer activity. These lipids were integrated into cubosomes, and the resultant structural modifications were linked to their molecular structure and concentration of the additive lipids, both in the presence and absence of serum. The ability of cationic cubosomes to kill cancer cells was investigated and was linked to both their inherent cytotoxicity and the cellular uptake of these lipids. Finally, the ability of these novel cationic cubosomes to co-deliver either a small molecule chemotherapeutic (paclitaxel) or siRNA was assessed. </p><p dir="ltr">Chapter 1 provides an overview of the literature relevant to the studies presented in this thesis. The chapter introduces the complex nature of cancer and the failure of some conventional treatment strategies. Modifications to conventional chemotherapy, including combination therapy involving molecular hybrids such as cationic lipid hybrids is described. An overview of common nanodrug delivery systems of prospective use in combination treatment, with simultaneous co-delivery of two or more drugs to the site of action is provided. The discussion focusses on lipid based nanocarriers as these are studied in the thesis. How the molecular architecture of the lipid dictates its self-assembly into a variety of lipid nanocarriers like liposomes, micelles and inverse bicontinuous cubic phases is described. Finally, a short review of cubosomes is presented, discussing their advantages in drug delivery, different formulation techniques, the phase behaviour of cubosomes in different environments and with various additives, surface functionalization of cubosomes, protein corona formation and its effect on cellular uptake behaviour, the cytotoxicity of cubosomes, and their pharmaceutical applications. </p><p dir="ltr">In Chapter 2, the evolution of the research field in the synthesis and characterisation of permanently charged cationic lipids is critically reviewed. The significance of molecular hybridization in crafting novel cationic lipids, characterized by a permanent positive charge in the headgroup region, is reviewed in depth. Preliminary structure-activity relationships are derived between the lipid structure (headgroup, spacer, hydrocarbon chain) and the self-assembled nanostructure adopted. In some cases, these structural features may be additionally linked to the intrinsic biological activity of the multifunctional cationic lipids in diverse applications in transfection, targeting and as therapeutic lipids or bioactive excipients. This review has been submitted to Small Science and is currently undergoing editor requested revision. </p><p dir="ltr">Chapter 3 focuses on DMP12, a bifunctional cationic lipid hybrid designed for targeted cancer therapy. DMP12 conjugates the potential pharmacophore 3-(3,4-dimethoxyphenyl) propionic acid to twin 12 carbon chain bearing a positively charged quaternary ammonium group. Encapsulation of DMP12 into cubosomes and the resulting changes in phase behaviour are assessed. The novelty of this study lies in exploring the potential of combination therapy, including the cytotoxic DMP12 and the small molecule therapeutic paclitaxel, against a paclitaxel-resistant prostate cancer cell-line. A synergistic effect was observed with co-delivery of DMP12 with paclitaxel achieving significantly higher levels of cell death, compared to control cubosomes loaded with either DMP12 or paclitaxel. While in vivo validation is essential, in vitro studies show that the rational design of DMP12 has led to improved loading efficiency in cubosomes and synergistic activity in combination with paclitaxel against resistant cancer cells. This study therefore highlights the potential of multifunctional cationic lipids in cancer therapy, particularly in combination with classical chemotherapeutics, resulting in selective cytotoxicity towards specific cancer lines while remaining hemocompatible. This chapter has been published in the Journal of Colloid and Interface Science (1). </p><p dir="ltr">In Chapter 4 the impact of the cationic lipid architecture on the phase behaviour of the cubosomes, their cellular uptake, and their cytotoxicity, particularly against cancer cells was investigated. Three novel cationic lipids, CCA12, ETD12, and U12 (each containing a pharmacophore linked to a twin 12 carbon chain bearing quaternary ammonium group) were designed and synthesized. The pharmacophore group was designed with increased size considerations resulting in lipids with systematically varying molecular architecture. All three lipids displayed a wedge-shaped molecular architecture with critical packing parameters (CPP) values expected to be >>1. The concentration-dependent phase transitions observed when these lipids were doped into MO cubosomes were consistent with the individual molecular architecture of each lipid. Transitions from the inverse bicontinuous primitive cubic (QIIP) phase adopted by pure monoolein cubosomes to more curved phases, such as inverse bicontinuous diamond cubic (QIID) and inverse hexagonal (HII) phases, were observed as the lipid concentration increased. For U12, the lipid containing the largest hydrophobic moiety, phase changes were observed at the lowest lipid loading. Compared to both the neutral lipid monoolein and the other cationic lipids (ETD12 and U12), CCA12 cubosomes displayed significantly higher uptake and cytotoxicity in a gastric cancer cell line (AGS). Cubosome zeta-potential was positively correlated with the AGS cell uptake. CCA12 cubosomes displayed the highest cytotoxicity against AGS cells, which was attributed to both the inherent cytotoxicity and high levels of cellular uptake. This work is in press at the Journal of Colloid and Interface Science.</p><p dir="ltr">Using multifunctional molecules or combining multiple molecules which can intervene with different pharmacological targets is considered as a promising strategy to treat cancer. In Chapter 5 cationic cubosomes were assessed for their ability to co-deliver both the cationic lipid CCA12 and the small molecule therapeutic paclitaxel to a prostate-resistant cancer cell?line DU-145. Results were compared to delivery of CCA12 and PTX individually and using a loose dose combination. The CCA12 lipid was observed to be hemocompatible and serum 5 albumin compatible. The concentration of the PTX affects the encapsulation efficiency; at higher PTX concentrations the encapsulation efficiency was generally lower. </p><p dir="ltr">Finally, in Chapter 5 the ability of CCA12 doped cubosomes to successfully deliver antisurvivin siRNA to PC-3 cells was assessed. Both gel retardation and DLS studies indicated that 2.5mol% CCA12 cationic cubosomes completely encapsulated the antisurvivin siRNA (15:1 ratio). A gene silencing study using reverse transcriptase-polymerase chain reaction (RT?PCR) indicated that the CCA12 cubosomes successfully transfected antisurvivin siRNA into PC-3 cell line and silenced the survivin levels in the cells. This early proof-of-concept study exemplifies the potential of cationic cubosomes as gene delivery carriers, with equivalent efficiency to the commercial transfection agent Lipofectamine. </p><p dir="ltr">Overall, the work presented in this thesis showcases the practical application of the multifunctional cationic lipids in the effective treatment of drug resistant prostate cancer cell lines in combination with the classical anticancer therapeutic, paclitaxel co-delivered using monoolein based cubosomes. This work is also an important step towards understanding the impact of the molecular architecture on the phase behaviour of the cubosomes which facilitates the rational design of cationic lipid excipients targeted to specific biomedical applications.</p>