Bibliographic record
Abstract
This thesis is focused on characterizing two lipid-based gene delivery systems: plasmid DNA-cationic lipid "complexes" and stabilized plasmid-lipid particles (SPLP). Complexes have utility for gene transfer in vitro whereas SPLP are designed for systemic gene therapy applications in vivo. In Chapter 2, the structural and fusogenic properties of complexes formed by mixing pCMV5 plasmid DNA with large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTMA) and l,2-dioleoyl-3-phosphatidylethanolamine (DOPE) or l,2-dioleoyl-3- phosphatidylcholine (DOPC) are examined and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between these cationic vesicles. Freeze fracture electron microscopy studies demonstrate association of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels. ³¹P NMR studies on the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" ³¹P NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader "bilayer" ³¹P NMR spectra. In agreement with previous studies, the transfection potency of the DOPE containing systems is dramatically higher than for the DOPC containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. It is suggested that the ³¹P NMR characteristics of complexes lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection. In Chapter 3, the influence of variations in the lipid component of SPLP on plasmid trapping and transfection potency in vitro are characterized. It is shown that SPLP formed with different monovalent cationic lipids exhibit similar plasmid entrapment properties but different transfection potencies. The poly(ethylene glycol) (PEG) density in SPLP can substantially influence both SPLP formation and transfection. By decreasing the length of the fatty acyl component of the PEG-ceramide anchor from 20 to 14 to 8 carbons, or by using smaller PEG chains (PEG₇₅₀, PEG₂₀₀₀ as compared with PEG₅₀₀₀), higher transfection levels were observed, consistent with a requirement for PEG removal in order for efficient transfection to occur. Further, it is shown that the primary factor limiting the transfection potency of SPLP is association and uptake into target cells. The final set of experiments in Chapter 4 was focused on characterizing the influence of the plasmid component in the formation of SPLP. It is shown that encapsulation efficiencies remain at 50 % or higher for (initial) plasmid-to-lipid ratios of up to 70 μg/μmol, allowing the proportion of lipid in empty vesicles following detergent dialysis to be significantly reduced compared to previous protocols. In addition, it is shown that the encapsulation efficiency is sensitive to the conformation of the plasmid employed, where higher encapsulation is observed for linearized plasmid as compared to plasmid in supercoiled or relaxed circular conformations. However, lower transfection potency for linearized plasmid was observed in SPLP and plasmid DNA-cationic lipid complexes.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".