Leech, Freya
(2024)
Evaluation of cationic lipid-based liposomes as a delivery vehicle for DNA therapeutics into dendritic cells.
PhD thesis, University of Nottingham.
Abstract
DNA vaccines have been licenced for various purposes, including a therapeutic canine melanoma vaccine (Grosenbaugh et al, 2011), a prophylactic equine west nile virus vaccine (Davis et al, 2001), and more recently the ZyCov-D SARS-Cov-2 vaccine for humans (Khobragade et al, 2022). DNA vaccines are stable, immunogenic, and provide a safe non-live approach to inducing both humoural and cellular immune responses in immune-rich environments such as the skin. Successful delivery of these vaccines is determined by the carrier since DNA alone fails to initiate immune responses due to rapid clearance in vivo. It was theorised that the formulation of a cationic liposome-DNA complex with controlled physicochemical characteristics would result in efficient uptake and expression of encoded proteins by dendritic cells (DCs), an essential step in initiating systemic immune responses.
Cationic lipids interact electrostatically with the negatively charged DNA backbone, resulting in a stable DNA-liposome complex with tunable characteristics. In this thesis, a well characterised liposomal formulation composed of DC-Cholesterol and DOPE was selected for initial evaluation. Liposomes were prepared by the thin film method at different molar ratios of DC-Cholesterol: 25 mol%, 30 mol%, 35 mol%, 40 mol%, and 50 mol%. Formulations were combined with DNA at nitrogen: phosphate (NP) ratios of 3, 6, 9, and 12. Hydrodynamic size, zeta potential, and encapsulation efficiency were all measured alongside morphology by cryogenic transmission electron microscopy and fusion studies using fluorescence resonance energy transfer. Liposomes containing 35 mol% DC-Cholesterol and higher formed stable complexes with 100% encapsulation efficiency regardless of NP ratio. To balance the need for stability, an overall positive complex charge, and DOPE content, liposomes composed of 40 mol% DC-Cholesterol were taken forward into transfection studies.
A panel of cell lines consisting of the human embryonic kidney cell line HEK293T, the murine fibroblast cell line NIH 3T3, and the murine dendritic cell line DC2.4 were selected for in vitro transfection studies. Liposomes were labelled with the lipophilic dye DiD and combined with a model plasmid encoding for green fluorescent protein and incubated with each cell line. Cells were stained with a live dead stain and assayed by flow cytometry. Uptake was evaluated by the percentage of live cells positive for DiD-associated fluorescence; more than 90% of live cells showed uptake, regardless of cell line or NP ratio. GFP expression was evaluated by measuring the percentage of live cells positive for GFP. Expression was dependent on both cell line and NP ratio, where the expression was highest in HEK293T cells, moderate in NIH 3T3 cells, and low in DC2.4 cells. Endosomal trapping was investigated as a potential mechanism limiting GFP expression by pretreating cells with chloroquine diphosphate, an endosomal disruptor. No difference in GFP expression was observed, either in population of cells positive for GFP or mean fluorescence intensity, suggesting endosomal trapping is not a limiting factor. In a balb/c murine vaccination study, in vivo immune responses to DC-Chol/DOPE liposomes containing 40 mol% DC-Cholesterol and a model SARS-Cov-2 pDNA combined at NP ratios of 3 and 9 were measured. Liposome doses were injected subcutaneously on days 1, 8, and 15 before being sacrificed on day 19. Antigen-specific serum antibody responses were measured by enzyme linked immunosorbent assay (ELISA) and splenocyte responses were measured by ELISpot. No immune responses were detected in response to liposomes formulated with DC-Cholesterol and DOPE.
It was subsequently hypothesised that exchanging DC-Cholesterol with the multivalent cholesterol based lipid GL67 would improve GFP expression in dendritic cells, and that doing so would correlate with better immune responses in vivo. It was also hypothesised that inclusion of linolenic acid, a fatty acid that has been shown to directly interact with dendritic cells along with its metabolites, may increase GFP expression. When linolenic acid was included with either DC-Chol/DOPE or GL67/DOPE, no difference in GFP expression was observed when compared to formulations without linolenic acid. Liposomes formed of 40 mol% GL67 and 60 mol% DOPE initiated strong expression of GFP in DC2.4 cells in a screening study, however, when taken forward into HEK293T and NIH 3T3 cells and three biological repeats carried out in all three cell lines, strong variation was observed in GFP expression. This may be linked to cell toxicity, cell variation, batch variation, temperature, or other unknown variables since the literature rarely describes both technical and biological replicability.
Overall, this study has demonstrated that multivalency is an important and so far underexplored factor in cationic lipid choice for liposomes for dendritic cell delivery. Liposomes formulated with the multivalent cationic lipid GL67 appeared to elicit stronger expression of GFP in DC2.4 cells than liposomes formulated with DC-Cholesterol, although variation between biological replicates impeded conclusive comparisons and highlighted a key gap in the literature in describing replicability. This provides key information for the future development of cationic liposome formulation for dermal dendritic cell transfection and vaccination.
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