The impact of size for liposomes modified with pH-responsive β-glucan derivatives on the initiation of cellular and humoral immune responses in murine models

Figures & data

Table 1. Characteristics of EYPC/chol (4/1, mol/mol) liposomes modified with MGlu-aquaβ.

Liposome	Pore size of membrane for extrusion	Size^a (nm)	PDI^a	Zeta potential (mV)	OVA/lipid^b (g/mol)
OL-NE	–	1425.4 ± 212	0.322	−43.6 ± 9.6	61.5 ± 13.6
OL-1000	1000 nm	756.3 ± 34.1	0.241	−52.0 ± 1.3	59.1 ± 4.5
OL-400	400 nm	316.4 ± 33.0	0.265	−38.8 ± 6.2	53.9 ± 8.1
OL-200	200 nm	148.2 ± 6.4	0.177	−54.6 ± 1.9	51.8 ± 9.5

^a Measured by dynamic light scattering.

^b Measured by Coomassie method for OVA-loaded liposomes modified with MGlu-Aquaβ.

OL-NE, non-extruded and OVA-loaded liposomes. OL-1000, OL-400, OL-200, OVA-loaded liposomes extruded through polycarbonate membranes of 1000, 400 or 200 nm pore sizes.

Figure 1. TNF-α secretion from DC2.4 cells treated with MGlu-Aquaβ-modified liposomes and TLR4 inhibition assay. DC2.4 cells were pre-incubated with or without TAK-242 (TLR4 signal inhibitor, 5 μmol/L). After 1 h incubation, the cells were treated with MGlu-Aquaβ-modified liposomes for 24 h (lipid concentration: 0.2 mmol/L). TNF-α in cell culture supernatants was measured using ELISA. OL-NE, non-extruded and OVA-loaded liposomes. OL-1000, OL-400, OL-200, OVA-loaded liposomes extruded through polycarbonate membranes of 1000, 400 or 200 nm pore sizes. Results denote the mean of triplicate experiments ± SEM. *p < .05, **p < .01, ***p < .001, ****p < .0001.

Figure 2. Cellular association of MGlu-Aquaβ-modified liposomes. DC2.4 cells were incubated with various size-controlled DiI-labeled liposomes modified with MGlu-Aquaβ (OL-NE, OL-1000, OL-400 or OL-200) for 4 h. The fluorescence intensities of the cells were analyzed using a flow cytometer. OL-NE, non-extruded and OVA-loaded liposomes. OL-1000, OL-400, OL-200, OVA-loaded liposomes extruded through polycarbonate membranes of 1000, 400 or 200 nm pore sizes. Results represent the mean values of triplicate experiments ± SEM.

Figure 3. Cytokine secretion in lymph nodes derived from mice immunized with PBS, OL-NE, OL-1000, OL-400 or OL-200. IFN-γ (a), TNF-α (b), IL-4 (c) and IL-12 (d) in lymph nodes collected 7 days after immunization were measured using ELISA. Each result represents the mean value ± SEM for a group of five mice. OL-NE, non-extruded and OVA-loaded liposomes. OL-1000, OL-400, OL-200, OVA-loaded liposomes extruded through polycarbonate membranes of 1000, 400 or 200 nm pore sizes. *p < .05, **p < .01.

Figure 4. Antitumor effects of OVA-loaded liposomes modified with MGlu-Aquaβ. Changes in the mice tumor volume (a) and survival of mice (b) were monitored after E.G7-OVA cell inoculation (5.0 × 10⁵ cells per mouse). C57BL/6 mice were immunized on days 7 and 14 after tumor inoculation with PBS and liposomes. Arrows indicate the days of sample injection. OL-NE, non-extruded and OVA-loaded liposomes. OL-1000, OL-400, OL-200, OVA-loaded liposomes extruded through polycarbonate membranes of 1000, 400 or 200 nm pore sizes. Each result represents the mean ± SEM for a group of five mice. *p < .05.

Figure 5. OVA-specific antibody production in BALB/c mice immunized with OVA-loaded liposomes modified with MGlu-Aquaβ. OVA-specific IgG1 (a) and IgG2a (b) titers in serum were measured using ELISA. Each result represents the mean value ± SEM for a group of four or five mice. *p < .05, **p < .01.

Data availability statement

All data needed to evaluate the conclusions in the article are present in the article and/or the Supplementary Materials. Additional data are available from the corresponding author [E.Y.] upon reasonable request.