A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer

Abstract

The immune checkpoint blockade therapy has profoundly revolutionized the field of cancer immunotherapy. However, despite great promise for a variety of cancers, the efficacy of immune checkpoint inhibitors is still low in colorectal cancer (CRC). This is mainly due to the immunosuppressive feature of the tumor microenvironment (TME). Emerging evidence reveals that certain chemotherapeutic drugs induce immunogenic cell death (ICD), demonstrating great potential for remodeling the immunosuppressive TME. In this study, the potential of ginsenoside Rg3 (Rg3) as an ICD inducer against CRC cells was confirmed using in vitro and in vivo experimental approaches. The ICD efficacy of Rg3 could be significantly enhanced by quercetin (QTN) that elicited reactive oxygen species (ROS). To ameliorate in vivo delivery barriers associated with chemotherapeutic drugs, a folate (FA)-targeted polyethylene glycol (PEG)-modified amphiphilic cyclodextrin nanoparticle (NP) was developed for co-encapsulation of Rg3 and QTN. The resultant nanoformulation (CD-PEG-FA.Rg3.QTN) significantly prolonged blood circulation and enhanced tumor targeting in an orthotopic CRC mouse model, resulting in the conversion of immunosuppressive TME. Furthermore, the CD-PEG-FA.Rg3.QTN achieved significantly longer survival of animals in combination with Anti-PD-L1. The study provides a promising strategy for the treatment of CRC.

Keywords: ATF6, activating transcription factor 6; ATP, adenosine triphosphate; CI, combination index; CRC, colorectal cancer; CRT, calreticulin; CTLA-4, cytotoxic T lymphocyte antigen 4; CXCL10, C-X-C motif chemokine 10; CXCL9, C-X-C motif chemokine 9; Chemotherapy; Colorectal cancer; Combination therapy; DAMPs, damage-associated molecular patterns; DCs, dendritic cells; ECL, enhanced chemiluminescence; EE, encapsulation efficiency; ER, endoplasmic reticulum; FA, folate; HMGB1, high-mobility group box 1; ICD, immunogenic cell death; IFN-γ, interferon-gamma; IL-10, interleukin-10; IL-12, interleukin-12; IL-4, interleukin-4; IL-6, interleukin-6; IRE1, inositol-requiring enzyme 1; Immunogenic cell death; Immunotherapy; LC, loading capacity; MDSCs, myeloid derived suppressor cells; MMR, mismatch repair; MR, molar ratio; NAC, N-acetyl-l-cysteine; NP, nanoparticle; Nano drug delivery system; PD-L1, programmed death-ligand 1; PEG, polyethylene glycol; PERK, PKR-like ER kinase; PFA, paraformaldehyde; PVDF, polyvinylidene fluoride; QTN, quercetin; ROS, reactive oxygen species; Reactive oxygen species; TAAs, tumor-associated antigens; TME, tumor microenvironment; Tumor microenvironment; UPR, unfolded protein response; p-IRE1, phosphorylation of IRE1; p-PERK, phosphorylation of PERK.

Graphical abstract

Figure 1

A combination of folate-targeted Rg3/QTN cyclodextrin-based co-formulation and anti-PD-L1 antibody for chemo-immunotherapy in CRC.

Figure 2

Ginsenoside Rg3 induced immunogenic cell death in CRC cells. (A) IC₅₀ of Rg3 for CT26 and HCT116 cells at 24 h. Data are presented as mean ± SD (n = 3). (B) Apoptosis (%) in CT26 and HCT116 cells following treatment of Rg3 at 6, 12 and 24 h. Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01 relative to DMSO. (C) The activity of UPR signaling pathways following treatment of Rg3 ([c] = 30 μmol/L) at 6, 12 and 24 h. The quantification was demonstrated in Fig. S1. (D) The characterization of ICD in CRC cells following treatment of Rg3 ([c] = 30 μmol/L), including CRT exposure (6 h), ATP secretion (12 h) and HMGB1 release (12 h). Data are presented as mean ± SD (n = 3). ∗P < 0.05 relative to DMSO; scale bar = 20 μm. (E) The expression of CD11c and CD86 in DCs stimulated with the supernatant from Rg3 ([c] = 30 μmol/L) -treated cells (24 h). Data are presented as mean ± SD (n = 3). ∗∗P < 0.01 relative to DMSO. (F) The in vivo vaccination assay using BALB/C and nude mice. Data are presented as mean ± SD (n = 4). ∗∗∗P < 0.001.

Figure 3

Quercetin caused reactive oxygen species in CRC cells. (A) IC₅₀ of QTN for CT26 and HCT116 cells at 24 h. Data are presented as mean ± SD (n = 3). (B) Apoptosis (%) in CT26 and HCT116 cells following treatment of QTN ([c] = 80 μmol/L) at 24 h. Data are presented as mean ± SD (n = 3). ∗∗P < 0.01 relative to DMSO. (C) The activity of Bcl-2/BAX/caspase 9/caspase 3 signaling pathways following treatment of QTN ([c] = 80 μmol/L) at 6, 12 and 24 h. The quantification was demonstrated in Fig. S2. (D) The ROS level in CT26 and HCT116 cells following treatment of QTN ([c] = 80 μmol/L) at 6, 12 and 24 h. Data are presented as mean ± SD (n = 3). ∗∗P < 0.01 and ∗∗∗P < 0.001 relative to DMSO. (E) Cell viability (%) of CT26 and HCT116 cells with or without NAC prior to treatment of QTN ([c] = 80 μmol/L) (24 h). Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01 relative to untreated control. (F) Apoptosis (%) in CT26 and HCT116 cells with or without NAC prior to treatment of QTN ([c] = 80 μmol/L) (24 h). Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01 relative to untreated control.

Figure 4

Synergistic effects of Rg3 and QTN in CT26 cells. (A) IC₅₀ of drug combination at 24 h. Data are presented as mean ± SD (n = 3). CI values at IC₅₀ were shown in Fig. S3. (B) Apoptosis (%) caused by drug combination at 24 h. Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01 relative to DMSO. (C) The CRT exposure with or without NAC before treatment of drug combination (6 h). Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01 relative to DMSO; scale bar = 20 μm. (D) The ATP secretion with or without NAC before treatment of drug combination at 12 h. Data are presented as mean ± SD (n = 3).∗P < 0.05 and ∗∗P < 0.01, between NAC and No NAC. (E) The HMGB1 release with or without NAC before treatment of drug combination at 12 h. Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, between NAC and No NAC. (F) The expression of CD11c and CD86 in DCs stimulated (24 h) by the supernatant from Rg3-treated cells with or without pretreatment of NAC. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, relative to DMSO.

Figure 5

Preparation and physicochemical characterization of FA-targeted co-formulation. (A) Formulation schematic. (B) The EE%, LC%, particle size and surface charge of targeted co-formulation. Data are presented as mean ± SD (n = 3). (C) TEM image of targeted co-formulation (scale bar = 100 nm). (D) The in vitro release of drugs from targeted co-formulation in 0.01 M PBS (pH = 5.5 and 7.4). Data are presented as mean ± SD (n = 4). (E) Particle size of targeted co-formulation following storage at 4 °C in aqueous solution. Data are presented as mean ± SD (n = 4). ∗P < 0.05 and ∗∗P < 0.01 relative to Day 0; NS, no significance. Non-targeted co-formulation also demonstrated similar physicochemical results observed by targeted counterpart.

Figure 6

In vitro studies of co-formulations. (A) Cellular uptake of co-formulations (NT co-formulation = non-targeted co-formulation; co-formulation = targeted co-formulation) containing Rhodamine was assessed at 6 h using confocal microscopy (scale bar = 5 μm). The quantification was shown in Fig. S4. (B) Cellular uptake of co-formulations containing Rhodamine was assessed at 4 h using flow cytometry (BD). Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, relative to PBS. (C) Cell viability (%) of co-formulations at 24 h. Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, relative to free drugs. (D) Cell-free areas before and after treatment of co-formulations (12 h) were imaged and measured for the relative scratch area (%) (scale bar = 50 μm). Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, relative to free drugs. (E) The colony formation following treatment of co-formulations (4 weeks) (scale bar = 50 μm). Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, relative to free drugs.

Figure 7

In vivo toxicity, pharmacokinetics and biodistribution of targeted co-formulation. (A) The body weight over a 30-day period following i.v. treatment of PBS and targeted co-formulation on Days 1, 3, 5, and 7. Data are presented as mean ± SD (n = 5). (B) Major organs were collected on Day 30 and assessed using H&E staining assay. No significant toxic sign was found in targeted co-formulation as compared to PBS (scale bar = 50 μm) (C) Hematological analysis including red blood cells (RBCs), white blood cells (WBCs), platelets (PLTs) and hemoglobin (HGB) was carried out on Day 30. Data are presented as mean ± SD (n = 4). (D) The liver/kidney functions including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CRE) were determined on Day 30. Data are presented as mean ± SD (n = 4). (E) The concentration of drugs in the plasma was plotted at different time points. Data are presented as mean ± SD (n = 4). (F) Biodistribution of DiD-labeled co-formulations was detected (640 nm/670 nm) using IVIS® In Vivo Optical System. Data are presented as mean ± SD (n = 4). ∗P < 0.05 in orthotopic CRC mouse model.

Figure 8

Combination therapy of targeted co-formulation and Anti-PD-L1 for CRC. (A) Treatment schedule and IVIS images. (B) The CRC progression over a 35-day period. Data are presented as mean ± SD (n = 5). ∗P < 0.05 and ∗∗P < 0.01; NS, no significance. (C) Animal survival (median survival: PBS ~38 days, Anti-PD-L1 ~40 days, targeted co-formulation ~62 days, and combination ≈ 96 days). Data are presented as mean ± SD (n = 5). ∗∗P < 0.01 and ∗∗∗P < 0.001. (D) Immunofluorescent staining assay (green = DNA fragments and blue = nuclei) on Day 20 to assess apoptosis in the tumor (scale bar = 50 μm). Data are presented as mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, relative to PBS. (E) Level of immune cells in the tumor on Day 20 was analyzed using flow cytometry (BD). Data are presented as mean ± SD (n = 4). ∗P < 0.05 and ∗∗P < 0.01; NS, no significance. (F) The mRNA expression of cytokines and chemokines in the tumor on Day 20 was analyzed using real time RT-PCR. Data are presented as mean ± SD (n = 4). ∗P < 0.05 and ∗∗P < 0.01; NS, no significance. (G) Orthotopic CRC mice treated with targeted co-formulation following the removal of CD4⁺ or CD8⁺ T cells. Data are presented as mean ± SD (n = 4). ∗P < 0.05 and ∗∗P < 0.01; NS, no significance.