Model biomimetic lipid membranes for encapsulation and controlled release of bioactive molecules in bicontinuous cubic phases
Creators: name: SARKAR, Sampa
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 340603
subject: Colloid and surface chemistry

description: Lipid-based nanovectors are of potential use in drug delivery and for gene therapy, due to their ability to enhance fusion with cellular membranes and transport drug molecules into the cytoplasm.  Lipidic materials are particularly important for the delivery of amphiphilic or hydrophobic biomolecules which are not amenable to traditional, solution-based delivery methods. They are biocompatible, protect the bioactive against degradation, and can offer controlled release properties.  This thesis investigates the use of lipid materials based on the bicontinuous cubic phase for the delivery of bioactive, including gene therapy and small molecule drugs. <br><br> 

Essentially all applications of bioactive-lipid hybrid materials require retention of functionality and accessible format. This is strongly influenced by the physical characteristics of the lipid matrix, which plays an important role in the functionality of bioactives which are embedded within it.  However, the majority of lipid vectors investigated to date have been formulated from a single lipid or a combination of two lipids in water. Such systems lack tunability, with only a narrow range of lattice parameters adopted. In addition, the lipid bilayer of these materials lacks the complexity of natural cell membranes, which are composed of hundreds of different lipids and which may be essential to retaining the functionality of bioactive embedded within them.<br><br>
In Chapter 2, the phase behaviour of complex quaternary lipid-water systems consisting of three different lipids (monoolein¿cholesterol¿phospholipid) and water was investigated using a combination of experimental and simulation techniques. This study provides a large library of lipidic materials with bilayer compositions, which more effectively mimic the native cell membrane and significantly increased tunability based on nanostructural parameters such as lattice parameter, aqueous channel size, and bilayer thickness. Extremely swollen cubic phases having a lattice parameter &gt; 300 Å were formulated. Unlike previously formulated swollen cubic phases these did not contain any charged lipids. They are therefore stable under physiologically relevant salt concentrations and are therefore particularly suitable for in vivo applications such as drug delivery. <br><br> 

In Chapters 3 and 4 the use of lipid-based nanovectors in gene therapy was explored due to their ability to enhance fusion with cellular membranes and transport the large polyanionic DNA molecules into the cytoplasm. In Chapter 3 a systematic analysis was carried out investigating the encapsulation and release of dsDNA fragments within cationic lipid phases of cubic symmetry (cationic cubic phases) and their dispersed sub-micron particles (cationic cubosomes).  Cationic cubic phases, both as the bulk phase and cubosomes, were formulated using six different cationic lipids (DOTAP, DODAB, DODAC, DDAB, CTAB and DODMA). The use of commercially available DNA ladders consisting of a controlled mixture of DNA fragments allowed us to determine release rates as a function of fragment size in a reasonably high-throughput manner. An improved understanding of the loading capacity and release profile of non-functional biomolecules in cationic cubosomes will assist in the design of novel lipid nanovectors for gene delivery.<br><br>

In Chapter 4 the cationic cubosomes characterised in Chapter 3 were used to deliver anti-sense GFP siRNA to GFP expressing CHO cells. Addition of a small amount of cationic lipid (0.1 mol%) resulted in complete loading of the anionic siRNA without significantly increasing toxicity; the fusogenic nature of the cubosomes was sufficient to drive the cellular interactions required for delivery.  Addition of siRNA resulted in only minor effects on the size and morphology of cuboplexes. The siRNA was successfully delivered to GFP expressing CHO cells with transfection efficiency higher than that of the commercially available lipofectamine. <br><br> 

In Chapter 5, cubosomes were investigated as delivery vehicles for TB drugs.   Five anti-TB drugs, Rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin were encapsulated in MO cubosomes.  Encapsulation of the anti-TB drug rifampicin in cubosomes improved the drug bioavailability in in vitro studies, with the time taken to eliminate the bacilli burden reduced from three to one day. Finally, a surface acoustic wave (SAW)-based device was used to nebulise drug-encapsulated cubosomes; retention of the internal nanostructure following nebulisation was confirmed. Future work will use in vitro and in vivo studies to assess the efficacy of traditional cubosome injections, and inhalation therapy using nebulised cubosomes, for the treatment of TB, including drug-resistant TB. <br><br>

This body of work constitutes a significant contribution to our understanding of the interactions between encapsulated biomolecules and the cubic phase bilayer. The structural trends and considerations highlighted have implications for the further implementation of these materials, including in the rational design and formulation of next generation gene and drug delivery materials.