Fig 1.
(A) Thin layer chromatography to show the conjugation of GP2 peptide to Maleimide-PEG-DSPE (1: GP2 peptide, 2:PEG2000-DSPE, 3: GP2-PEG2000-DSPE). (B)SDS-PAGE analysis (1: liposome, 2: GP2, 3: GP2-PEG2000-DSPE, 4: Lip-GP2, 5: Ladder).(C) Chromatographic analysis of GP2 peptide and the conjugated peptide-PEG-DSPE, included subset graphs are the HPLC monitoring of free peptide as a reference denoted by (-) and the conjugated peptide was determined post reaction with Maleimide-PEG2000-DSP denoted by (…).
Table 1.
Characteristics of liposomal formulations (n = 3; Mean ±SD).
Fig 2.
Splenocyte cell phenotype and level of cytokine expression (A and B) and lymph nodes (C) of BALB / c mice immunized with different liposomal formulations. 14 days after the last immunization, splenocytes were isolated and stimulated in vitro with PMA/I for 4h and stained with a surface CD8 and CD4 marker and intracellular IFN-γ and IL-4 cytokine prior to FACS analysis. (A)Geometric mean fluorescence intensity (MFI) level for INF-γ in gated CD8 and CD4 in the spleen. (B)MFI level for IL-4 in gated CD4 lymphocyte populations in the spleen. (C) MFI level for INF-γ in gated CD8, CD4, and IL-4 in gated CD4 in lymph nodes. Data represent mean± SEM (= 3).*p<0.05, **p<0.01, and ***p<0.001; denote significant difference from all other formulations and the HEPS-dextrose 5%.
Fig 3.
Induction of rHER2/neu peptide-specific intracellular cytokine response in splenocytes as determined by the ELISpot assay.
Mice were immunized with three booster doses of 10 μg/mice using different liposomal formulations. Two weeks after the last injection, splenocytes from three mice from each group were harvested and re-stimulated withGP2 peptide (A). Immune responses were evaluated with IFN-γ ELISpot assay kit and (B) IL-4 ELISpot assay kit. The data indicate the mean ±SEM(n = 3).*p<0.05, and ***p<0.001; denote significant difference from all other formulations and the HEPS-dextrose 5%.
Fig 4.
In vitro antigen-specific CTL response in different formulations for splenocytes isolated from vaccinated mice.
CTL response was assessed by Calcein AM-loadedrHER2/neu-expressing TUBO cells and rHER2/neu negative CT26 cells, Data are shown as mean ± SEM (n = 3). **p<0.01, and ***p<0.001; denote significant difference from the HEPS-dextrose 5%.
Fig 5.
Analysis of IFN-γ and IL-4 levels in splenocytes isolated from BALB/c mice vaccinated with Lip-DOPE-MPL-GP2, two weeks after the final vaccination. All values represent means ± SD (n = 3).
Fig 6.
Prophylactic effects of vaccination in BALB/c mice against a TUBO tumor model.
Two weeks after the last booster, five mice in each group were challenged subcutaneously with 5 ×105 TUBO cells. Mice were observed for tumor growth (A) and survival (B). Tumor size was calculated based on three dimensions, two times every week. The survival of mice was followed for about 75 days. The data indicate the mean± SEM (n = 5). **p<0.01, ***p<0.001, and **** p<0.0001; denote significant difference from the HEPS-dextrose 5% group.
Table 2.
Prophylactic efficacy data of different liposomal vaccine formulations in TUBO tumor model of mice (n = 5).
Fig 7.
Therapeutic effects of liposomal formulations in BALB/c mice against a TUBO tumor model.
Two weeks after inoculation of 5 ×105 TUBO cells to five mice in each group, different liposomal formulations were administrated three times with two-week intervals. After the first injection, the mice were challenged and tumor size was calculated based on three dimensions. (A) Tumor growth was measured two times every week. (B) The survival of mice was followed for 91 days. The data indicate the mean ±SEM(n = 5). ***p<0.001 and ****p<0.0001; denote significant difference from the HEPS-dextrose 5% group.
Table 3.
Therapeutic efficacy data for liposomal formulations in TUBO tumor mice model (n = 5).