Remodeling tumor microenvironment using pH-sensitive biomimetic co-delivery of TRAIL/R848 liposomes against colorectal cancer

Abstract

Background: Despite significant advancements in the development of anticancer therapies over the past few decades, the clinical management of colorectal cancer remains a challenging task. This study aims to investigate the inhibitory effects of cancer-targeting liposomes against colorectal cancer.

Materials and methods: Liposomes consisting of 3β-[N-(N', N'-dimethylamino ethane)carbamoyl]-cholesterol (DC-CHOL), cholesterol (CHOL), and dioleoylphosphatidylethanolamine (DOPE) at a molar ratio of 1:1:0.5 were created and used as carriers to deliver an apoptosis-inducing plasmid encoding the tumor necrosis factor-related apoptosis-inducing ligand (pTRAIL) gene, along with the toll-like receptor (TLR7) agonist Rsiquimod (R848). The rationale behind this design is that pTRAIL can trigger cancer cell apoptosis by activating the DR4/5 receptor, while R848 can stimulate the immune microenvironment.

Results: Experimental results demonstrated the synergistic effects of R848 and pTRAIL encapsulated by liposomes (RTL) in suppressing the proliferation of colorectal cancer cells. Moreover, further in vivo investigations revealed the strong anti-tumor efficacy of RTL in xenograft and orthotropic in situ models of colorectal cancer.

Conclusions: These findings collectively highlight the therapeutic potential of R848/pTRAIL-loaded liposomes in the treatment of colorectal cancer.

Keywords: Colorectal cancer; Plasmid TRAIL (pTRAIL); R848; Tumor-associated macrophages.

Figure 1. Characterizations of liposomes. (A) DLS measurement of particle size distribution of liposomes consisting of DC-CHOL, CHOL and DOPE. (B) A representative SEM image of liposomal particles. Scale bar: 200 nm. (C) Particle size changes of liposomes in PBS (pH = 7.4) containing 10% FBS after 72 h incubation at 37°C, and (D) for 30 days at 4°C. (E) The in vitro release profile of R848-encapsulated liposomes at various intervals in both the acidic (pH = 6.5) and neutral (pH = 7.4) environments. The data are expressed as the mean ± SD, n = 3.

Figure 2. Zeta potential values (surface charge potential) were determined in blank liposomes, R848 liposomes, pTRAIL liposomes and the R848/pDNA (RTL) liposomes. Data are expressed as the mean ± SD; n = 3; **p < 0.01; ***p < 0.001.

Figure 3. Uptake and transfection assay of cationic pDNA/liposome. (A) CT26 cells were incubated with liposomes containing GFP-expressing pDNA for 1 or 4 h. After 24 h, GFP signal fluorescence was detected. Scale bar: 100 µm. (B and C) The liposomal delivery efficiency of pDNA was compared to the PEI2.5K transfection reagent. Liposomes containing pDNA with the luciferase reporter gene were used to incubate the cells, and PEI2.5K transfection reagent was used as the positive control. The negative control (NC) represented the cells without any treatment, while CNTL represented the cells which were incubated with free pDNA only. The delivery efficiencies of pDNA in different experimental groups were measured in (B) CT26 cells and (C) HCT116 cell line based on the luciferase activity. (D) Representative images of cells incubated with pDNA liposome or pDNA/PEI2.5K transfection complex. Data are expressed as the mean ± SD; n = 3; ***p < 0.001; Scale bar: 200 µm.

Figure 4. The in vitro anti-cancer effects of liposomal formulations. (A) HCT116 cells were treated with different concentrations of free R848 and R848 liposomes for 48 h, and a CCK8 cell viability assay was conducted to compare cell viability. (B) Cell viability analysis in HCT116 cells treated with free R848, R848-encapsulated liposome, pTRAIL-loaded liposome and pTRAIL/R848 liposome (RTL) at a concentration of R848 = 10 μM for 48 h. (C) Cell viability analysis in HCT116 cells treated with different concentrations of blank liposomes for 48 h. (D) Cell viability analysis of HCT116 cells treated with R848/pTRAIL liposomes (RTL) at different R848 concentrations (pTRAIL at 1 µg/mL). (E) Representative images of calcein AM-PI staining in control and RTL-treated HCT116 cells. Scale bar: 300 µm. Data are expressed as the mean ± SD; n = 3; *p < 0.05; **p < 0.01; ***p < 0.001 compared with CNTL group. ^###p < 0.001 compared with free R848 group.

Figure 5. The effects of liposome treatment on the EMT biomarkers. Human monocyte THP-1 cells were polarized into M2-like macrophages and then co-cultured with HCT116 cells in the presence or absence of RTL for 48 h. qRT-PCR analysis was conducted to detect the relative expression levels of (A) E-cadherin, (B) N-cadherin, (C) Vimentin, (D) Snail, (E) Slug, and (F) Twist in HCT116 cells. Data are expressed as the mean ± SD; n = 3; ***p < 0.001.

Figure 6. The effects of liposome treatment in a xenograft model of HCT116 cells. (A) Schematics of the xenograft tumor model establishment of HCT116 cells in nude mice. The tumor-bearing mice were randomly divided into 4 groups: (i). the control group (treated with empty liposome), (ii). the RL group (treated with R848 liposome), (iii). the pTRAIL group (treated with pTRAIL liposome), and (iv). the RTL group (treated with RTL liposome). (B) Tumor volume growth record in each group. (C) Tumor weight summary in each group. (D) TUNEL staining of apoptotic cell death in the tumor samples of each group. Scale bar: 1000 µm. Data are expressed as the mean ± SD; n = 5 animals in each group; **p < 0.001; ***p < 0.001 compared with CNTL group. ^###p < 0.001 compared with RTL group.

Figure 7. Effects of liposome treatments on cytokines in an orthotopic in situ model of CT26 cells. (A) The schematic illustration showing an orthotropic in situ model of CT16 cells at the colon tissue. The mice were divided into five groups: the healthy group (no tumor cell inoculation); the cancer model group (CT26 cell inoculation); the cancer model +R848 liposome treatment group; the cancer model+pTRAIL liposome treatment group; and the cancer mdoel+RTL treatment group. (B) The summary of animal survival in each experimental group. (C) The summery of body weight changes in each experimental group within 20 days after tumor cell inoculation. (D) TGF-β levels, (E) MIF levels, and (F) IFN-γ levels in tumor tissues of each experimental groups were detected by ELISA. Data are expressed as the mean ± SD; n = 10; **p < 0.01; ***p < 0.001.