3.1 Preparation and characterization of the nanoparticles
Due to the presence of protonated amino groups in the CH, negatively charged siRNA can be loaded through electrostatic interaction. The CH/siRNA nanoplexes can effectively interact with cell membranes. Therefore, we developed the CH nanoparticles to deliver HIF-1α siRNA into tumor cells. The CH/HIF-1α siRNA nanoplexes were prepared by incubation of the nanoparticles with siRNA. The physical structure of the nanoparticles and the CH/siRNA nanoplexes was confirmed by DLS. The size, PDI, and zeta potential of the nanoparticles were 254 ± 7 nm, 0.4 ± 0.06, and 17.7 ± 0.7 mV, respectively (Fig. 1A). The mean diameter of the CH/siRNA nanoparticles was 243 ± 6 nm with the size distribution being relatively narrow (0.3 ± 0.04). The siRNA binding to the nanoparticles and formation of the CH/siRNA nanoplexes was also evident as the zeta potential of nanoparticles changed to 12.2 ± 0.4 mV. These results have been reported in previous studies [31]. To find the proper ratio of the nanoparticles and siRNA (N/P) reaching the maximum encapsulation, the gel retardation assay was performed. We prepared the CH/siRNA nanoplexes at the different ratios of N/P (20 to 60) in gel electrophoresis. The results indicated that full complexation was conformed at an N/P = 60 ratio (Fig. 1B).
3.2 Cytotoxicity of the nanoparticles in CT26 cells
To evaluate the cytotoxic activity of the nanoparticles against CT26 cells, the MTT assay was performed on the cells treated with the nanoparticles for 48 h. Figure 1C shows cell viability after incubation with various concentrations of the nanoparticles in the range of 10–100 µg/ml. Compared to the non-treated cells, the viability of cells incubated with the nanoparticles was 85%, indicating good biocompatibility and low cytotoxicity of the nanoparticles.
3.5 In vitro HIF-1α gene-silencing by CH/HIF-1α siRNA nanoplexses
The gene silencing efficiency of CH/ HIF-1α siRNA nanoparticles was initially evaluated in the cell culture. The relative HIF-1α mRNA levels were evaluated in CT26 cells incubated with CH/siRNA in different molarities of HIF-1α siRNA (25, 50, 75, and 100 nM) at N/P = 60 ratios. Untreated cells and free siRNA were considered as controls. Gene expression was analyzed after 48 h by qPCR. As shown in Fig. 1D, the HIF-1α mRNA levels in 100nM siRNA significantly reduced in comparison with other concentrations and the control group (P < 0.0001).
3.6 Animal model study
3.6.1 Synergistic effects of combination therapy on tumor growth inhibition
The therapeutic effects of combination therapy were assessed by s.c. challenging BALB/c mice with CT26 tumor cells, followed by the drug administration of OXA, IMQ, and HIF-1α siRNA based on the treatment protocol (figure 2A). All mice tolerated treatment with OXA, IMQ, and HIF-1α siRNA in the administrated dose and scheduled treatment (established by literature review). To evaluate the effect of combination therapies on tumor growth inhibition, we initially investigated their effects on tumor phenotypes in the mice. Figure 2B shows the inhibitory effects of combination therapy on tumor size and volume.
While tumor volume was strikingly increasing in untreated mice, treatment of a dual or complete mixture of HIF-1α siRNA, OXA, and IMQ significantly suppressed tumor growth. The co-administration of HIF-1α siRNA+ OXA significantly inhibited tumor growth. The simultaneous administration of IMQ with HIF-1α siRNA or OXA gradually induced significant tumor shrinkage from day 1 through day 10 compared with PBS or CH groups. The triple combination therapy with HIF-1α siRNA+ OXA+ IMQ significantly inhibited the growth of established tumors in CT26 tumor-bearing mice (Figure 2C) (P < 0.0001) but did not induce any complete tumor regression. In comparison, tumor volume between dual and triple combination therapies showed that only chemoimmunotherapy with IMQ and OXA was statistically significant (p = 0.004).
Combination therapy diminished the average tumor weight in all treatment groups. The average tumor weights decreased in mice exposed to IMQ with HIF-1α siRNA or OXA compared with the PBS and the nanoparticles groups (Figure 2D). Notably, the combination of HIF-1α siRNA+ OXA, as well as triple combination therapy, resulted in a further shrinkage of tumor weight compared with the control group (p< 0.0001). Our combination therapy gradually decreased tumor growth from day 6 to the final observation day (day 10), and the mean tumor volume was significantly reduced (Figure 2E). In total, the tumor weights in the combination treatment groups were 52% lower than those in the control one, which was much lower than those in the HIF-1α siRNA+ OXA+ IMQ (12.3%) and HIF-1α siRNA+ OXA (17.7%) groups. In comparison, tumor weight between dual and triple combination therapy groups showed that co-treatment of IMQ with HIF-1α siRNA or OXA was statistically significant. Our results demonstrated that HIF-1α siRNA and OXA administration efficiently reduced tumor growth, and combined treatment with IMQ exerted additional effects in CT26 tumor models.
3.6.2 The effects of the CH/HIF-1α siRNA nanoplexes on HIF-1α down-regulation in CT26 syngeneic mice model
We investigated the inhibition effects of i.t. injections of the CH/HIF-1α siRNA nanoplexes on the expression level of HIF-1 on tumor samples compared with control. As shown in Figure 3A, the relative HIF-1α mRNA level in tumor tissue with an injection of the CH/HIF-1α siRNA nanoplexes was markedly reduced in comparison to uninjected groups. The results of qPCR performed on day 10 revealed a 70% decrease in the HIF-1α mRNA and protein levels in HIF-1α siRNA-treated tumor tissue compared with the other groups.
3.6.3 The effect of combination therapy on apoptosis stimulation
To further elucidate the impact of the combination therapy on tumor cells, we studied expression levels of apoptosis-related genes in CT26 tumor samples. The mRNA expression level of pro-apoptotic genes such as BAD and BAX significantly increased in mice treated with HIF-1α siRNA+ OXA. While the expression level of Bcl-2 as an anti-apoptotic gene increased in the control groups, in mice treated with triple combination and OXA+ IMQ significantly decreased (Figure 3B, C, and D). The BAX protein expression level significantly increased in mice treated with a triple combination therapy group in comparison with other groups. Our combination treatment exerted direct apoptosis stimulatory effects.
3.6.4 The effect of combination therapy on CT26 tumor cell proliferation
The effect of the combination treatment was also evaluated on cell proliferation. For the initial assessment, we investigated signal transducer and activator of transcription 3 (STAT3) and vascular endothelial growth factor (VEGF) gene expression profiles in the tumor tissue specimens after treatment based on the protocol. The mRNA expression level of STAT3 and VEGF genes significantly decreased in all mice in treated groups compared with PBS and the nanoparticles group (Figure 4A and B). Analysis of protein expression levels showed that the STAT3 protein in mice treated with OXA + IMQ significantly decreased (Figures 4C). Our data showed that the STAT3 gene expression in mice treated with a triple combination was significantly lower compared with the dual combination. These data led us to hypothesize that the triple combination induces further downregulation of cell proliferation genes, leading to greater inhibition of tumor growth.
3.6.5 Combination therapy induces immune cell-related cytokines and Th1 polarization in the TME
The immune-related effects of the combination treatment was assessed through analysis of the mRNA and protein level of cytokines between treated and untreated tumor samples. In comparison with dual combination therapy, triple combination strongly upregulated Th1(T helper1)-type cytokines including IL-12 (Interleukin-12) and IFN-γ, but not Th2-type cytokines such as IL-4 and IL10 (Figure 5 A, B, C, and D). These results showed that the co-administration of HIF-1α siRNA+ OXA +IMQ induces cellular immune responses in tumor-bearing mice. In the dual treatment groups, no significant differences were observed in the mRNA expression levels of IL12 and IL4 cytokines compared to the control group. Analysis of the protein expression levels showed that the IFN-γ in mice treated with the triple combination significantly increased compared with control (Figure 5E). Altogether, triple combination therapy had a strong local effect on the TME and polarizes it towards an inflammatory, Th1 cytotoxic immune contexture which may directly inhibit tumor growth.
4.6 Correlation of HIF-1α gene expression with CT26 tumor growth phenotypes
After the preliminary analysis, we tested the correlation between HIF-1α gene expression and tumor size in all tumor sample groups. The pattern of the genetic correlation coefficient is shown in Figure 6. The positive correlation coefficient (r=0.68) was obtained between tumor size and HIF-1α expression level in all groups (p= 0.003) (Figure 5A). In the triple combination therapy group, HIF-1α had positive correlations with VEGF and tumor size although they were not statistically significant (r=0.8) (Figure 5C). The expression level of BCL2 and IL-12 genes had negative correlations with HIF-1α expression. The most significant genetic correlation (p=0.09) was between HIF-1α and BCL2 genes. The relationships between the HIF-1α gene and other studied genes were simple because a low level of HIF-1α in TME is often associated with a strong immune response and tumor growth inhibition.