Developing lipid-based nanoparticles for applications in synthetic biology and nanomedicine

Non-lamellar lipid lyotropic liquid crystal nanoparticles (LCNPs) formed by inverse type lipids have internal nanostructures that possess 2 or 3-dimensional periodicity. These internal nanostructures are based on the inverse bicontinuous cubic (VII), or 2D-hexagonal (HII) phases or ordered inverse micellar phases such as the inverse micellar cubic phases (III) which are termed cubosomes, hexosomes and inverse micellar cubosomes respectively. In recent years there has been an increasing interest in using LCNPs in (a) nanomedicine because LCNPs contain versatile and tuneable internal nanostructures with high surface area for manipulation of drug loading, targeting and triggered release, and (b) synthetic biology due to their ability to generate spontaneous curvature on the membrane. The simultaneous interplay of phenomena and enormous complexity of living systems makes the understanding of living systems incredibly difficult, including the understanding of the interaction between LCNPs and cells, even for simple minimal cells. The vital functions of cell membranes are based on their ability to quickly shape and transform the plasma membrane to perform multiple tasks such as growth, division, endocytosis and apoptosis. Proteins and lipid compositions mainly modulate the morphological transformation of membrane in vivo through very complex processes. Thus, the redesign and engineering of biomimic systems that are simple enough to interrogate various biological behaviours to simulate entirely novel functions has emerged as an ambitious challenge into the field of synthetic biology. Also, the study of interaction between LCNPs and model membrane can help to assist the LCNPs clinical application. In this dissertation, LCNPs were developed for applications in nanomedicine and synthetic biology.
There is a growing demand to develop stimuli-responsive smart nanocarriers which have potential to provide enhanced dose, temporal, and spatial control over compounds and chemical processes. The naturally occurring pH gradient found throughout the body makes pH an attractive stimulus for guiding nanocarrier responses to specific locations or (sub)cellular compartments in the body. Firstly, we designed highly sensitive LCNPs using monoolein (MO), oleyl alcohol (Olalc) and DOBAQ that reversibly respond to changes in pH by changing the connectivity within their structures at physiological temperatures. At pH 7.4, the internal structure of the nanoparticles consists of discontinuous inverse micellar ‘water pockets’ based on the space group Fd3m. At pH ≤6, the nanoparticles changed from a closed internal structure to an accessible open tubular internal structure based on a two-dimensional inverse hexagonal phase (planar group p6mm). The internal structure of the nanoparticles was determined using small-angle X-ray scattering and cryogenic transmission electron microscopy. The obtained high-resolution electron microscopy images allowed for the first time to directly visualize the internal structure of Fd3m nanoparticles and resolve two reverse micelles of different sizes, which constitute the structural motifs within the Fd3m unit cell and compare them to theoretical geometric models. Moreover, this is the first demonstration of a pH-induced phase transition from a compartmentalized to an accessible porous structure upon lowering the pH,
To foster the application of LCNPs in nanomedicine, the understanding of their cytotoxicity is of significance. The effect of LCNPs with different internal nanostructures (VII, HII and III) was further investigated with respect to cytotoxicity. A thorough investigation of the nanoparticles was conducted using a variety of experimental techniques such as X-ray scattering, cryo-TEM and dynamic light scattering. MTT assays were also performed to understand the in vitro cytotoxicity of the LCNPs. HII and III were found to be more toxic than VII against three epithelia mammary cell lines (1-7HB2, MDA-MB-468, and MCF10A). The live cell images suggested that these LCNPs might interact with cells via different uptake mechanisms. These findings might contribute to the understanding of fundamental steps for engineering of LCNPs in further clinical development.
Finally, the interaction between LCNPs and giant unilamellar vesicles (GUVs), a cell-size model membrane, were investigated and an analogous membrane remodelling process seen in real cells is presented that can be induced by MO based LCNPs. Data showed that LCNPs successfully incorporate into DOPC GUV membranes. The different curvature of lipids on the membrane allows the generation of spontaneous curvature and further induce different morphological and topological change. Using real-time fluorescence confocal microscopy, various life-like dynamic events in GUVs are observed including swelling, tubulation, fission, fusion, budding and intraluminal vesicles formation induced by LCNPs. The change of permeability of the membrane was also investigated.