A lipid/PLGA nanocomplex to reshape tumor immune microenvironment for colon cancer therapy

Abstract

Immune checkpoint blockade therapy provides a new strategy for tumor treatment; however, the insufficient infiltration of cytotoxic T cells and immunosuppression in tumor microenvironment lead to unsatisfied effects. Herein, we reported a lipid/PLGA nanocomplex (RDCM) co-loaded with the photosensitizer Ce6 and the indoleamine 2,3-dioxygenase (IDO) inhibitor 1MT to improve immunotherapy of colon cancer. Arginine-glycine-aspartic acid (RGD) as the targeting moiety was conjugated on 1,2-distearoyl-snglycero-3-phosphoethanolamine lipid via polyethylene glycol (PEG), and programmed cell death-ligand 1 (PD-L1) peptide inhibitor DPPA (sequence: CPLGVRGK-GGG-d(NYSKPTDRQYHF)) was immobilized on the terminal group of PEG via matrix metalloproteinase 2 sensitive peptide linker. The Ce6 and 1MT were encapsulated in PLGA nanoparticles. The drug loaded nanoparticles were composited with RGD and DPPA modified lipid and lecithin to form lipid/PLGA nanocomplexes. When the nanocomplexes were delivered to tumor, DPPA was released by the cleavage of a matrix metalloproteinase 2-sensitive peptide linker for PD-L1 binding. RGD facilitated the cellular internalization of nanocomplexes via a_vβ₃ integrin. Strong immunogenic cell death was induced by ¹O₂ generated from Ce6 irradiation under 660 nm laser. 1MT inhibited the activity of IDO and reduced the inhibition of cytotoxic T cells caused by kynurenine accumulation in the tumor microenvironment. The RDCM facilitated the maturation of dendritic cells, inhibited the activity of IDO, and markedly recruited the proportion of tumor-infiltrating cytotoxic T cells in CT26 tumor-bearing mice, triggering a robust immunological memory effect, thus effectively preventing tumor metastasis. The results indicated that the RDCM with dual IDO and PD-L1 inhibition effects is a promising platform for targeted photoimmunotherapy of colon cancer.

Keywords: IDO inhibition; colon cancer; immune checkpoint blockade; lipid/PLGA nanocomplex; photodynamic therapy.

Graphical abstract

Figure 1.

Illustration of lipid/PLGA nanocomplex activated antitumor immune effect through multiple synergistic strategies.

Figure 2.

TEM images of CM (A), RCM (B) and RDCM (C); scale bars represent 50 nm. DLS sizes of blank CM, RCM and RDCM (D). The release curves of Ce6 (E) and 1MT (F) in vitro. Cell viability of CT26 cells incubated with 1MT, CM-1, RCM-1 and RDCM-1 (G), Ce6 + 1MT, CM, RCM and RDCM for 48 h (H). Cell viability of CT26 cells incubated with Ce6 + 1MT, CM, RCM and RDCM for 24 h after 660 nm laser irradiation for 30 s (I) (n = 3, *P < 0.05, **P < 0.01).

Figure 3.

CLSM images and FCM results of cellular uptake (A, C and D) and intracellular ROS (B, E and F) (n = 3, *P < 0.05, **P < 0.01). Apoptosis rates of cells treated with Ce6 + 1MT, CM, RCM and RDCM after 660 nm laser irradiation (G).

Figure 4.

CLSM images and semi-quantitative of CRT (A, C) and HMGB1 (B, D) of CT26 cells treated with Ce6 + 1MT, CM, RCM and RDCM after 660 nm laser irradiation. ATP released from CT26 cells treated with Ce6 + 1MT, CM, RCM and RDCM after 660 nm laser irradiation (E). Affinity detection of RDCM to PD-L1 after pretreatment with MMP-2 (F and G). Kyn inhibition rates of CT26 cells (H). n = 3, *P < 0.05, **P < 0.01.

Figure 5.

Tumor volumes (A), body weights (B), tumor weights (C), tumor inhibition rates (D) of mice in different groups, n = 5, *P < 0.05, **P < 0.01. FCM analysis of CD11c and CD80 expression analysis (G), CD11c and CD86 expression analysis (H), and statistical results of CD11c⁺ CD80⁺ cells (E) and CD11c⁺ CD86⁺ cells (F), n = 5, *P < 0.05, **P < 0.01. Statistical results of percentages of CTLs in T cells (I), percentages of Tregs in T cells in different treatment (J), ratios of CTLs/Treg T cells (K), n = 4, *P < 0.05, **P < 0.01. The ratio of Trp/Kyn (L) in the serum of mice. CD8 immunofluorescence analysis of the tumor slices in different groups (M), n = 4, *P < 0.05, **P < 0.01.

Figure 6.

Scheme of immune memory study (A), in vivo bioluminescence images at different time points and representative intestinal bioluminescence imaging at day 21 of mice in different treatment groups (B). Body weight of mice during treatment (C). Number of intestine nudes (D), FCM analysis of T_EM in spleens (gated on CD3⁺ CD8⁺ T cells) (E) (n = 4, *P < 0.05, **P < 0.01).