Cationic DOTAP/DC-chol liposome elicits dose-dependent OVA-specific antibody production depending on antigen (A) and liposome (B) concentrations.

<p>BALB/c female mice were immunized intranasally with various doses of OVA plus DOTAP/DC-chol liposomes on days 0 and 7. Serum was collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay. The data are obtained from at least three independent experiments and are expressed as the mean ± the standard error. Significance was assessed with the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i><0.05, **<i>p</i><0.01.</p

<i>In vitro</i> antigen-specific production of IFN-γ and IL–4 in splenocytes and nasal passages from BALB/c mice immunized intranasally with OVA and DOTAP/DC-chol liposomes.

<p>Splenocytes from vaccinated BALB/c mice were cultured for 72 h in the presence of OVA (0, 1, 10, or 100 μg/ml). After culture, the supernatants were collected, and concentrations of IFN-γ and IL–4 in the culture supernatants were determined by ELISA assay. The data are representative of at least three independent experiments and are expressed as the mean ± standard deviation for samples assayed in triplicate. Significance was assessed with the <i>t</i>-test with Welch correction: *<i>p</i><0.05.</p

Kinetics of the appearance of OVA-specific serum IgG in BALB/c female mice immunized intranasally with OVA and DOTAP/DC-chol liposomes.

<p>BALB/c female mice were immunized intranasally with PBS alone, DOTAP/DC-chol liposomes (0.4 μmol/mouse) alone, OVA (5 μg/mouse) alone, or OVA (5 μg/mouse) plus DOTAP/DC-chol liposomes (0.4 μmol/mouse) once weekly (days 0, 7, 14, 21, and 28). Serum was collected every week immediately prior to immunization (days 0, 7, 14, 21, 28, and 35). Anti-OVA IgG, IgG1, and IgG2a levels in serum were determined by ELISA assay. The data are obtained from at least three independent experiments and are expressed as the mean ± the standard error. Significance was assessed with the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i><0.05, **<i>p</i><0.0001.</p

Role of liposome surface charge on the mucosal adjuvant effect.

<p>BALB/c female mice were immunized intranasally with PBS, OVA (5 μg/mouse) alone, OVA (5 μg/mouse) plus DOTAP/DC-chol liposomes (0.4 μmol/mouse), anionic PS liposomes (0.4 μmol/mouse), or neutral PC liposomes (0.4 μmol/mouse) on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay. The data are obtained from at least three independent experiments. The box-plot shows the median value with the 25th-75th percentiles and the error bars indicate the 5th-95th percentiles. Significance was assessed using the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i><0.01.</p

Induction of OVA-specific serum IgG and nasal tissue IgA responses in BALB/c mice immunized intranasally with OVA and cationic liposomes.

<p>BALB/c female mice were immunized intranasally with PBS, OVA (5 μg/mouse) alone, or OVA (5 μg/mouse) plus various cationic liposomes (0.4 μmol/mouse) on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay as described in the “Materials and Methods” section. The data were obtained from at least three independent experiments. The box-plot shows the median value with the 25th-75th percentiles and the error bars indicate the 5th-95th percentiles. Significance was assessed using the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i> < 0.05, **<i>p</i> < 0.01, NS: not significant.</p

Assessment of the <i>in vivo</i> safety of DOTAP/DC-chol liposomes in mice by body weight loss and gene expression of an inflammatory cytokine, TNF-α, at the site of delivery.

<p>(A) BALB/c female mice were immunized intranasally with PBS, DOTAP/DC-chol liposomes (0.4 μmol/mouse) alone, or OVA (5 μg/mouse) plus DOTAP/DC-chol liposomes (0.4 μmol/mouse) once per week. Body weight was recorded over 21 days. Significance was assessed using a two-way repeated measures ANOVA with Bonferroni post–hoc test: NS: not significant, (B) Sixteen hours after the immunization, nasal tissues were collected and the expression of TNF-α mRNA was quantified by semi-quantitative RT-PCR. Densitometry values for mRNA were normalized to β-actin as an internal standard. The data are expressed as the mean ± standard deviation for samples assayed in triplicate. Significance was assessed using <i>t</i>-test with Welch correction: NS: not significant.</p

Comparison of the mucosal adjuvant activity of DOTAP/DC-chol liposomes with CT.

<p>BALB/c female mice were immunized intranasally with PBS, OVA (5 μg/mouse) alone, OVA (5 μg/mouse) plus DOTAP/DC-chol liposomes (0.4 μmol/mouse), or OVA (5 μg/mouse) plus CT (1 μg/mouse) on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay as described in the “Materials and Methods” section. The data were obtained from at least three independent experiments. The box-plot shows the median value with the 25th-75th percentiles and the error bars indicate the 5th-95th percentiles. Significance was assessed using the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i> < 0.05, NS: not significant.</p

Influence of DOTAP/DC-chol liposome particle size on the mucosal adjuvant effect.

<p>BALB/c female mice were immunized intranasally with PBS, and treated with DOTAP/DC-chol liposomes of various particle sizes (0.4 μmol/mouse) alone, OVA (5 μg/mouse) alone, or OVA (5 μg/mouse) plus DOTAP/DC-chol liposomes of various particle sizes (0.4 μmol/mouse) on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay. The data are obtained from at least three independent experiments. The box-plot shows the median value with the 25th-75th percentiles and the error bars indicate the 5th-95th percentiles. NS: not significant as assessed by the Kruskal–Wallis with Dunn’s post–hoc test.</p