Fig 1.
Structural schematic of conventional surfactants & gemini surfactants (A), and chemical structure of 16-3-16 gemini surfactant (B).16-3-16 Properties: cmc = 0.026 mM32, Krafft Temperature = 42°C 39.
Fig 2.
One-step in vivo LCC DNA minivector production system.
The in vivo production system involves a recombinant E. coli for thermoregulated expression of Tel protelomerase. In the temperature inducible system, protelomerase expression is repressed by a CI[Ts]857 repressor at temperatures below 37°C. Temperature upshift to 42°C causes instability and dissociation of the thermolabile repressor which allows for controlled expression of protelomerase. Subsequent enzymatic activity of the expressed protelomerase on parental pDNA vector substrates results in DNA processing into LCC DNA ministrings.
Fig 3.
Parental plasmid DNA vector substrate for the production of LCC DNA ministrings.
The 5.6 kb pDNA vector (pNN9) possesses two "Super Sequences" (SS) flanking the eukaryotic expression cassette for the generation of LCC DNA ministrings. Within each Super Sequence, pal act as the protelomerase recognition sequences for the production of LCC DNA ministrings (2.4 kb) upon processing by Tel protelomerases. SV40 enhancer sequences serve as DNA-targeting sequences (DTS) for improved nuclear entry during gene delivery.
Table 1.
Generation of CCC pDNA and LCC DNA ministring-derived lipoplexes.
Table 2.
Particle size & zeta potential of CCC pNN9, LCC DNA ministring, 16-3-16, and DOPE.
Table 3.
Particle size and zeta potential of DNA/16-3-16 and DNA/16-3-16/DOPE lipoplexes.
Fig 4.
Particle size variations for DNA/16-3-16/DOPE lipoplexes with increasing N+/P− charge ratios.
As lipoplexes approach charge neutralization, a significant increase in particle size led to highly variable particles conferring large aggregate formation. Large aggregates for LCC/16-3-16/DOPE and CCC/16-3-16/DOPE lipoplexes appeared most prominent at charge ratios of 1:1 and 2:1 respectively. Progressive decreases to particle sizes at higher charge ratios led to stable and uniform particle formation.
Fig 5.
DNase sensitivity assay for DNA/16-3-16 and DNA/16-3-16/DOPE lipoplexes.
DNA ladder (lane 1); pNN9 (CCC) (lane 2); DNase I exposed pNN9 (CCC) (lanes 3 & 4); CCC/16-3-16 lipoplexes at 2:1, 5:1, and 10:1 charge ratios (lanes 5–7); CCC/16-3-16/DOPE lipoplexes at 2:1, 5:1, and 10:1 charge ratios (lanes 8–10); 16-3-16/DOPE (lane 11); DNA ministring (LCC) (lane 12); DNase I exposed DNA ministring (LCC) (lanes 13 & 14); LCC/16-3-16 lipoplexes at 2:1, 5:1, and 10:1 charge ratios (lanes 15–17); LCC/16-3-16/DOPE lipoplexes at 2:1, 5:1, and 10:1 charge ratios (lanes 18–20). Equal loading of the recovered DNA solution after DNase I exposure indicated improved DNA recovery for ministring (LCC) derived lipoplexes. DNA/16-3-16 lipoplexes conferred better DNA recovery after DNase I degradation over DNA/16-3-16/DOPE lipoplexes across all of the tested charge ratios.
Fig 6.
DNase sensitivity assay for A) CCC/16-3-16/DOPE lipoplexes and B) LCC/16-3-16/DOPE lipoplexes.
DNA ladder (lane 1); DNA (lane 2); DNase I exposed DNA (lane 3); DNA/16-3-16/DOPE lipoplexes at 2:1 charge ratio (lanes 4 & 5); DNA/16-3-16/DOPE lipoplexes at 5:1 charge ratio (lanes 6 & 7); DNA/16-3-/16/DOPE lipoplexes at 10:1 charge ratio (lanes 8 & 9). Equal loading confirmed improved DNA recovery after DNase I exposure at higher charge ratios. A majority of the complexed DNA ministrings (LCC) was recovered in lipoplexes at 5:1 and 10:1 charge ratios.
Fig 7.
Effect of DNA topology on in vitro gemini surfactant-based transgene delivery.
Parent CCC plasmid (pNN9) DNA vectors were processed into LCC DNA ministrings by passing through the one-step heat-inducible mini DNA vector production system. 0.4 μg of each respective DNA vector was mixed with gemini surfactant 16-3-16 at charge ratios of 5:1 or 3:1 in presence or absence of helper lipid, DOPE, and transfected into human-derived ovarian cancer (OVCAR-3) cells. Cells were collected 48 h post-transfection and analyzed by flow cytometry for green fluorescent protein (eGFP) expression (A) and cytotoxicity of synthetic carriers using red propidium iodide (PI) fluorescent protein (B). Transfection efficiency was measured as the number of eGFP-expressing cells divided by the total number of cells. PI was added to assess transfection associated cytotoxicity. All data were expressed by GraphPad as mean ±SEM. Statistical differences were determined using a two-way ANOVA test followed by Bonferroni's post-tests. Stars indicate significantly higher transfection efficiencies of LCC DNA ministrings compared to pNN9 parent plasmid (P ≤ 0.001).