Self-Crosslinking Lipopeptide/DNA/PEGylated Particles: A New Platform for DNA Vaccination Designed for Assembly in Aqueous Solution

Abstract

Delivery of plasmids for gene expression in vivo is an inefficient process that requires improvement and optimization to unlock the clinical potential of DNA vaccines. With ease of manufacture and biocompatibility in mind, we explored condensation of DNA in aqueous solution with a self-crosslinking, endosome-escaping lipopeptide (LP), stearoyl-Cys-His-His-Lys-Lys-Lys-amide (stearoyl-CH₂K₃), to produce cationic LP/DNA complexes. To test whether poly(ethylene glycol) (PEG)-ylation of these cationic complexes to neutralize the surface charge would improve the distribution, gene expression, and immune responses poly(ethylene glycol), these LP/DNA complexes were combined with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀). Fluorescence imaging illustrated that the cationic complexes exhibited the highest degree of localization and lowest degree of dispersion throughout the injected muscle, suggesting impaired mobility of cationic particles upon administration. Nanoluciferase reporter assays over a 90-day period demonstrated that gene expression levels in muscle were highest for PEGylated particles, with over a 200-fold higher level of expression than the cationic particles observed at 30 days. Humoral and cell-mediated immune responses were evaluated in vivo after injection of an ovalbumin expression plasmid. PEGylation improved both immune responses to the DNA complexes in mice. Overall, this suggests that PEGylation of cationic lipopeptide complexes can significantly improve both the transgene expression and immunogenicity of intramuscular DNA vaccines.

Keywords: DNA vaccine; PEGylation; gene expression; humoral and cell-mediated immunity; intramuscular; lipopeptide; non-viral gene delivery.

Graphical abstract

Figure 1

Structures of the LP and PEGylated Lipid Used to Form DNA Complexes Stearoyl-CH₂K₃ lipopeptide as C-terminal amide (A) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀) (B).

Figure 2

Characterization of the LP/DNA Complexes (A) Dye exclusion profile of LP/DNA complexes prepared with stearoyl-CH₂K₃. All data points are calculated as the percentage of fluorescence intensity of plasmid DNA in solution. *p < 0.05 and ****p < 0.0001 for significant differences in fluorescence intensity compared with DNA alone (one-way ANOVA, Dunnett’s post hoc test). (B and C) Zeta potential (B) and mean particle size and polydispersity index (C) of stearoyl-CH₂K₃/DNA complexes over a range of charge ratios. As the (+/−) charge ratio of LP to DNA increased, the zeta potential increased, whereas the Z-average remained low (generally between 10–100 nm), with the exception of complexes formed at charge ratios close to unity. All data are presented as mean ± SEM of n = 3 separate experiments. (D and E) Cryo-TEM images of the LP/DNA complexes prepared at a charge ratio of (D) 1.5:1 and (E) 2.5:1, illustrating aggregate complexes (denoted by the large white circle) and smaller, ∼30-nm particles (denoted by the small white circle), respectively. Scale bars, 100 nm (D) and 50 nm (E).

Figure 3

Characterization of the DSPE-PEG₂₀₀₀/LP/DNA Complexes (A and B) PEGylated LP/DNA complexes prepared at various charge ratios and the resultant (A) zeta potential and (B) particle size and polydispersity index. A reduction in zeta potential was observed with addition of negatively charged DSPE-PEG₂₀₀₀ to the LP/DNA complex. Data are presented as mean ± SEM, n = 3. (C and D) Cryo-TEM images of the DSPE-PEG₂₀₀₀/LP/DNA complexes at (C) pre-ultrafiltration and (D) post-ultrafiltration (5,000 × g for ∼5.5 hr at 4°C) with Amicon Ultra-15 (NMWL of 3 kDa). Filled-in white arrowheads denote example DSPE-PEG₂₀₀₀/LP/DNA particles, and the white arrow outline denotes free uncondensed plasmid. Scale bars, 100 nm.

Figure 4

Particle Stability on Addition of NaCl and Relative Gene Expression in Muscle after Intramuscular Injection (A) The mean particle size and polydispersity index of LP/DNA prepared at a charge ratio of 2.5:1 and DSPE-PEG₂₀₀₀/LP/DNA prepared at a charge ratio of 0.75:2:1 with and without addition of 0.15M NaCl. *p < 0.05 for the increase in Z-average in comparison with the untreated control (paired Student’s t test). Data are presented as mean ± SEM, n = 3. (B) Relative gene expression determined as relative light units (RLUs) in the calf muscle 1, 30, and 90 days after injection of DNA. Data are presented as mean ± SEM of n = 3–6 mice. **p < 0.01 and ****p < 0.0001 for significant expression levels between treatments as indicated (two-way ANOVA, Tukey’s post hoc test). Background RLUs determined in saline-treated controls were less than 1.

Figure 5

Local Distribution of DNA Complexes In Vivo (A) Total target plasmid detected by qPCR in the injected calf muscle 30 min after injection of naked DNA, LP/DNA, or DSPE-PEG₂₀₀₀/LP/DNA. Data are presented as mean ± SEM of n = 4 mice. (B) Total DNA pixel count in the muscle of naked DNA-AF594-, LP/DNA-AF594-, or DSPE-PEG₂₀₀₀/LP/DNA-AF594-injected mice at 30 min. Data are presented as mean ± SEM of n = 3 mice, with a total of 12 muscle sections (10 μm, every 800 μm) collected from each mouse. *p < 0.05 for significant differences in pixel count (one-way ANOVA, with Tukey’s post hoc test). (C–N) Example sections of skeletal muscle imaged after injection of saline (C–E), naked DNA-AF594 (F–H), LP/DNA-AF594 (I–K), or DSPE-PEG₂₀₀₀/LP/DNA-AF594 (L–N). Autofluorescence from tissue to illustrate muscle morphology is shown in green and AF594-labeled plasmid in red. The images in (C)–(E) were captured with a 4× objective lens.

Figure 6

Immunogenicity of DNA Complexes (A) Ovalbumin-specific antibody activity levels in sera of mice (relative to the positive index) after administration of DNA complexes or control formulations. Symbols represent the individual data points, whereas the line represents the mean. The dotted line denotes the positive index of 1. Values higher than 1 are positive for anti-OVA activity; values less than 1 are negative for anti-OVA activity. (B) Percentage of target epitope-specific cells eliminated in mice immunized with OVA-coated splenocytes (positive control), saline (negative control), LP/DNA, or DSPE-PEG₂₀₀₀/LP/DNA. Data are presented as the mean ± SEM. **p < 0.01 and ****p < 0.0001 for significant activity levels compared with saline-treated mice (one-way ANOVA with Dunnett’s post hoc test). (C–E) Representative histograms showing the distinct OVA_257–264 pulsed (high CFSE concentration) and unpulsed (low CFSE concentration) population of splenocytes detected in mice immunized with saline (C), LP/DNA (D), or DSPE-PEG₂₀₀₀/LP/DNA (E) formulations.