Fig 2: Frequency of tumor-infiltrated lymphocytes (TILs) and T cell subpopulations in the tumor environment of different groups. Two days after the last injection at the fourth week, three mice from each treatment group were sacrificed and cells from tumor tissue were isolated. Isolated cells were stained with conjugated antibodies against surface markers (CD3, CD4, CD8, and CD25 FoxP3). FACS analysis was carried out to determine the frequency of lymphocytes secreted into the tumor site. (A) The frequency of TIL following injection of mice with different formulations. (B) Liposomal formulation alone or in combination with GE11-Lipo CD73 siRNA decreased the frequency of CD3/CD4 positive lymphocytes. (C) Lipo Dox formulation group significantly decreased the frequency of CD3/CD8 positive lymphocytes (P value < 0.01). (D) Lipo Dox formulation negatively affected the frequency of CD8+, INF+ lymphocytes. In combination group, the negative effect of Lipo Dox formulation is attenuated by GE11-Lipo CD73 siRNA formulation. (E) Frequency of CD4+, INF+ in CD3 gated lymphocytes. (F) Effect of different formulation on the frequency of Treg CD25+, Foxp3+ lymphocytes. Data are reported as mean ± SD (n = 3). Statistically significant differences are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: Morphology of empty liposomes (A, B) and liposomes Containing CD73 siRNA (C, D) by atomic force microscopy (AFM). Both empty liposomes and liposomes containing CD73 siRNA have spherical shape with high resolution and an average size of less than 100 nm. Figure (A) and (C) show the two-dimensional (2D) image of empty liposome and liposomes containing CD73 siRNA respectively. Figure (B) and (D) show the Three-dimensional (3D) images of empty liposome and liposomes containing CD73 siRNA respectively.

Fig 4: Intracellular cytokine profile of tumor-infiltrated lymphocytes (TILs). Two days after the last injection at the fourth week, three mice from each treatment group were sacrificed and tumor tissue cells were isolated. Isolated cells were stained with intracellular markers. Fluorescence intensity (geometric mean) of cytokines including IL-4, IFN-γ, and FoxP3 were assessed by flow cytometry. (A, C) Different formulations had not any significant differences on the geometric mean of IFN and IL-4 in the CD4+ gated cells. (B) Injection of Liposomal siRNA, GE11targeted siRNA-loaded liposomes and the combination group (GE11-Lipo CD73 siRNA+ Lipo Dox) markedly increased the geometric mean of IFN-γ in CD8+ T cells. (D) On the other hand, these treatments significantly decreased the florescent intensity of FoxP3 compared to control (PBS) and Liposomal doxorubicin groups. Data are reported as mean ± SD (n = 3). Statistically significant differences are represented as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Efficacy of liposomal formulations on both CD73 gene expression and CD73 ectonucleotidase level by RT-PCR and flow cytometry, respectively, in the tumor tissue of mice inoculated with 4T1 cell line. (A) The result of RT-PCR showed that the targeted form of liposome containing CD73 siRNA (GE11-Lipo CD73 siRNA) alone and in combination with Liposomal doxorubicin could efficiently reduce the expression of CD73 gene (****: P value < 0.0001). (B) Decreased fluorescence intensity indicates decreased CD73 ectonucleotidase level. Histogram shows the intensity of PE fluorescence in the treatment groups. CD73 PE G mean stands for geometric mean of fluorescence intensity of CD73 PE. Data are shown as the mean ± SD (n = 3). Statistically significant differences are shown as follow: **P < 0.01 and ****P < 0.0001.