1346242-81-6 br As shown in Fig B survival analysis up to
As shown in Fig. 5B, survival analysis (up to 32 days) indicated the 4. Discussion slowest tumor growth in PBS group. The log-rank (Mantel-cox) survival analysis revealed no statistically diﬀerences between the survival rates In the current study, four common liposomal platforms used in
of the mice groups. cancer immunotherapies were investigated in terms of cytotoxicity,
Fig. 4. CD4+ T and CD8+ T lymphocytes frequencies as well as subtypes of T lymphocytes, i.e. Th1, Th2, and Treg were measured using flow cytometry. A represents respectively the lymphocyte gating in terms of CD4 cell marker followed by the quadrants of IFN- γ and IL-4-producing and non-producing cells, the lymphocyte gating in terms of CD8 class followed by quadrant of IFN- γ producing and non-producing cells, and the quadrant of Treg based on the cell markers of CD25-FITC and Foxp3 PE. None of the liposome formulation changed the frequency of CD4+ cell frequency (B), CD4+ IFN- γ- (C) and CD4+ IL-4-producing 1346242-81-6 (D). The liposome formulations also did not change the frequency of CD8+ cell frequency (E). However, F1 and F2 significantly increased the proportion of the CD8+ IFN-γ-producing cells as opposed to others, including the control PBS (F). None of the liposomes changes the frequency of Treg cell population (G). Data are presented as individual values and mean (n = 5). Statistically significant diﬀerences are shown as follows: **p < 0.01, ***p < 0.001.
Fig. 5. Therapeutic eﬀect of the liposomal formulations in BALB/c mice bearing C26 colon carcinoma. Seven days after inoculation of C26 cells, when the dimension of tumor reached about 3 mm3, all mice (five mice in each group) were treated with liposomal formulations. None of the formulation exerted therapeutic eﬀect in terms of tumor growth restriction (A) and event-free survival (B). The data are shown as the mean ± SEM (n = 5) for tumor size analysis and percent survival for survival analysis.
Therapeutic eﬃcacy indices of liposomal formulations in mice bearing C26 tumor.
Treatment group MSTa(day) Average of TTEb TGD (%)d ILS (%)e
a Median survival time.
b Time to reach end point.
d Tumor growth delay.
e Increase life span.
immunogenicity and therapeutic properties. The liposome ingredients (lipids) are varied in chemical structure, molecule net charge and crystalline-to-gel phase transition temperature or Tm. HSPC with the primary lipid component of DSPC and DSPG are solid at the body temperature, given their Tm = 55 0C, while DMPC, DMPG, DOTAP and DOPE have respectively Tm of 24, 23, 4, and −11 °C. Accordingly, it could be inferred that F1- and F2 liposomes had fluid membrane in the body, while F3- and F4 liposomes were solid assemblies in the body (Anderson and Omri, 2004; Chen et al., 2013).
Moreover, it is determined that the liposomes of diﬀerent size, surface charge, membrane stability and composition exhibited varied level of immunogenic responses as well as to immune cell polarization (Brewer et al., 1998). In the current study, the liposomes exhibited si-milar particle size distribution (given the Z-average and PdI parameters in Table 1); however, they were diﬀerent in terms of liposome net surface charge and membrane fluidity. It was found out that the posi-tively charged F1-DOTAP- and DOPE-containing liposome was the most eﬀective liposomal formulation to induce immune system in our dif-ferent immunogenic assessments of the liposomes. F1-L induced the serum lymphocytes to produce IL-4 and IFN-γ and splenocytes to se-crete these cytokines. It also polarized the lymphocyte population to-ward CD8+ CTL- IFN-γ –producing cells.
At the second place, it was F2-DOPE-containing liposome that produced the abovementioned results as for F2-L, which is surmised to have fluid membrane at body temperature (37 °C). In contrast, the corresponding F3-DOPE-containing liposome displayed no im-munogenic activity or immune system polarization as with the PBS control and F4-PEGylated liposomes. It is imagined that substitution of DMPC and DMPG lipids with the respective DSPC and DSPG lipids re-sults in a liposomal membrane with high stability at the body tem-perature and low tendency to induce lymphocytes, thereby leading to F3-L immune inactivity as opposed to the corresponding F1-L.
DOTAP and DOPE lipids, as well as other lipids in these categories, are shown to enhance the transfection eﬃciency to immune and non-immune cells and they are utilized as lipid helpers. On the other hand, it is found that cholesterol, which is known to stabilize the liposome membrane, reduces the transfection eﬃciency (Ramezani et al., 2009). Whether or not a corresponding F3-choesterol-free liposome formula-tion could exhibit improved immune activity as compared to the pre-sent F3-liposome formulation remains an unanswered question in the current study.