A smart dual-drug nanosystem based on co-assembly of plant and food-derived natural products for synergistic HCC immunotherapy

Abstract

Nanotechnology has emerged as an ideal approach for achieving the efficient chemo agent delivery. However, the potential toxicity and unclear internal metabolism of most nano-carriers was still a major obstacle for the clinical application. Herein, a novel "core‒shell" co-assembly carrier-free nanosystem was constructed based on natural sources of ursolic acid (UA) and polyphenol (EGCG) with the EpCAM-aptamer modification for hepatocellular carcinoma (HCC) synergistic treatment. As the nature products derived from food-plant, UA and EGCG had good anticancer activities and low toxicity. With the simple and "green" method, the nanodrugs had the advantages of good stability, pH-responsive and strong penetration of tumor tissues, which was expected to increase tumor cellular uptake, long circulation and effectively avoid the potential defects of traditional carriers. The nanocomplex exhibited the low cytotoxicity in the normal cells in vitro, good biosafety of organic tissues and efficient tumor accumulation in vivo. Importantly, UA combined with EGCG showed the immunotherapy by activating the innate immunity and acquired immunity resulting in significant synergistic therapeutic effect. The research could provide new ideas for the research and development of self-assembly delivery system in the future, and offer effective intervention strategies for clinical HCC treatment.

Keywords: Aptamer; Co-assembly; EGCG; HCC; Immunotherapy; Nanodrug; Natural product; Synergistic treatment; Ursolic acid.

Graphical abstract

Figure 1

Schematic design and treatment of the dual-drugs co-assembly nano-delivery system. (A) The preparation of the “carrier-free” Apt-modified nanodrug based on the UA and EGCG. (B) Synergistic HCC treatment of the nanosystem by activating the innate and acquired immunity.

Figure 2

Characterization of prepared co-assembly NPs. (A) Mean sizes and zeta potential of UEA NPs. (B) Polyacrylamide gel electrophoresis of Apt, UE and UEA NPs to make sure the conjugation of Apt to NPs. (C)‒(D) AFM and TEM images of UA and UEA NPs.

Figure 3

Cytotoxicity assay of UA and NPs in cancerous cells and noncancerous cells. Inhibitory effects on (A) HEK293T, L02 cells and (B) HepG2, HeLa cells after incubated with different concentrations of UA, UA NPs, UE NPs, and UEA NPs for 24 h. Data are presented as mean ± SD (n = 6); ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Figure 4

Cellular uptake of UEA NPs in HepG2 cells and HeLa cells. Confocal images of HepG2 cells (A) and HeLa (B) incubated with UA, UA NPs, UE NPs and UEA NPs for 4 h. (C)‒(D) Flow cytometry detection of cell uptake and mean fluorescence intensity of HepG2 and HeLa cells after incubation with different formulations. Data are presented as mean ± SD (n = 3); ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Figure 5

Investigation of in vivo antitumor effect of the nanosystem. (A) Tumor weight excised from mice after different formulations of treatment at 21 days. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 significant in comparison to the NS control group. (B) Image of tumor tissues separated from mice treated with different formulations. (C) Tumor volume growth curves after different formulations of treatment. The difference between UEA NPs and other groups were statistically significant (∗∗P < 0.01, ∗∗∗P < 0.001). (D) Tumor inhibition ratio of different formulations in HCC treatment. ∗∗∗P < 0.001 significant in comparison to the NS control. (E) HE staining of heart, liver, spleen, lung and kidney from mice of WT and UEA NPs.

Figure 6

In vivo fluorescence imaging of free Cy5 and different of NPs. (A) Fluorescence images of HCC model mice injected with three NPs recorded at 0.5, 2, 6, 12, and 24 h with the free Cy5 as control. (B) Fluorescence images of the major organs and tumors of HCC model mice injected with free Cy5 and NPs. (C)‒(D) The average fluorescence intensity of tumor site at different time points, the major organs and tumors of different groups. Values represented are the mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 7

Assessment of immunity activation of co-assembly UEA NPs. (A) Schematic illustration of the induced immunotherapy of UA and EGCG in UEA NPs. (B) Relative mRNA expression of cytokines secretion in mouse PBMC after treated with UA, UA NPs, UE NPs, UEA NPs at the same concentration. ∗∗P < 0.01, ∗∗∗P < 0.001 significant in comparison to the control. (C)‒(D) Effect of UA, EGCG, Apt and NPs groups on expression of CD4⁺ and CD8⁺ T cells in vivo. ∗P < 0.05; ∗∗∗P < 0.001 significant in comparison to the NS group. (E) Serum IL-12 and IFN-γ levels post intravenous administration of 0.9% saline, UA NPs, UE NPs or UEA NPs. ∗P < 0.05; ∗∗∗P < 0.001 significant in comparison to the NS group. (F) Tumor-infiltrating CD8⁺ T cells from tumor tissues. Data are presented as mean ± SD (n = 5); ∗P < 0.05; ∗∗∗P < 0.001.