Nanoparticle/Engineered Bacteria Based Triple-Strategy Delivery System for Enhanced Hepatocellular Carcinoma Cancer Therapy

Abstract

Background: New treatment modalities for hepatocellular carcinoma (HCC) are desperately critically needed, given the lack of specificity, severe side effects, and drug resistance with single chemotherapy. Engineered bacteria can target and accumulate in tumor tissues, induce an immune response, and act as drug delivery vehicles. However, conventional bacterial therapy has limitations, such as drug loading capacity and difficult cargo release, resulting in inadequate therapeutic outcomes. Synthetic biotechnology can enhance the precision and efficacy of bacteria-based delivery systems. This enables the selective release of therapeutic payloads in vivo.

Methods: In this study, we constructed a non-pathogenic Escherichia coli (E. coli) with a synchronized lysis circuit as both a drug/gene delivery vehicle and an in-situ (hepatitis B surface antigen) Ag (ASEc) producer. Polyethylene glycol (CHO-PEG₂₀₀₀-CHO)-poly(ethyleneimine) (PEI_25k)-citraconic anhydride (CA)-doxorubicin (DOX) nanoparticles loaded with plasmid encoded human sulfatase 1 (hsulf-1) enzyme (PNPs) were anchored on the surface of ASEc (ASEc@PNPs). The composites were synthesized and characterized. The in vitro and in vivo anti-tumor effect of ASEc@PNPs was tested in HepG2 cell lines and a mouse subcutaneous tumor model.

Results: The results demonstrated that upon intravenous injection into tumor-bearing mice, ASEc can actively target and colonise tumor sites. The lytic genes to achieve blast and concentrated release of Ag significantly increased cytokine secretion and the intratumoral infiltration of CD4/CD8⁺T cells, initiated a specific immune response. Simultaneously, the PNPs system releases hsulf-1 and DOX into the tumor cell resulting in rapid tumor regression and metastasis prevention.

Conclusion: The novel drug delivery system significantly suppressed HCC in vivo with reduced side effects, indicating a potential strategy for clinical HCC therapy.

Keywords: anti-angiogenesis; chemotherapy; engineered bacteria; hepatocellular carcinoma; immunotherapy.

Graphical abstract

Figure 1

Quorum-induced release of Ag by engineered immunotherapeutic bacteria encoding a SLC. (A) E. coli with SLC reach a quorum and induce the phage-lysis protein X174E, leading to bacterial lysis and release of a constitutively produced. E. coli growth dynamics over time of SLC⁺ (B) and SLC⁻ (C) E. coli in liquid culture (n = 3). (D) Ag concentration of the supernatant after Ag release E. coli cultured for 8 h. (****P < 0.0001).

Figure 2

Characterization of ASEc@PNPs. (A) TEM image (inset) and size distribution of PNPs. (B) DOX relative loading of the ASEc. (C) Bacterial viability of ASEc@PNPs at different DOX concentrations. (D) TEM images of E. coli and PNPs-loaded ASEc. Scale bar: 1 μm. (E) CLSM image of PNPs-loaded ASEc. Red fluorescence represents PNPs, and blue fluorescence represents DAPI labeled ASEc. Scale bar: 10 μm. (F) Hydrodynamic diameter and (G) zeta potential of ASEc, and PNPs -loaded ASEc. (H) In vitro drug release curves of ASEc@PNPs.

Figure 3

In vitro analysis of ASEc@PNPs. (A) Cytotoxicity of the ASEc, PNPs (no hsulf-1), PNPs and ASEc@PNPs. (B) CLSM images of HepG2 cells incubated with ASEc, PNPs (no hsulf-1), PNPs and ASEc@PNPs. Scale bar: 100 μm. Green and red represent live cells and dead cells, respectively. Cell uptake of PNPs and ASEc@PNPs through (C) CLSM images and (D) flow cytometry. Scale bar: 25 μm. Transfection of PNPs through (E) FCM and CLSM images. Scale bar: 50 μm.

Figure 4

Hsulf-1 suppresses migration and angiogenesis. (A) Wound healing assays of HepG2 cells. Scale bar: 100 μm. Histograms show relative cell migration (B). Hsulf-1 inhibits VEGF-induced tube formation of HUVECs (C and D). Scale bar: 25 μm.

Figure 5

ASEc@PNPs induced the ICD of tumor cells and promoted macrophage polarization in vitro. (A) FCM analysis of the proportion of M1 macrophages (labeled with CD86⁺). Cytokine content of (B) TNF-α and (C) IL-6 tested by ELISA. (D) Immunofluorescence staining images of HepG2 cells. Scale bar: 10 μm. (E) Mean fluorescence intensity of CRT tested by FCM. (**p < 0.01, ***p < 0.001, ****p < 0.0001). Scale bar: 10 μm.

Figure 6

In vivo biodistribution of ASEc@PNPs. (A and B) E. coli colony numbers and pictures of plates of major organs and tumor tissues after being homogenized and incubated at 37 °C for 24 h (n = 3). (C) Ex vivo images of major organs and tumors at various time points after ASEc and ASEc@PNPs intravenous injection. (D) Immunofluorescence images of tumor sections intravenously injected with ASEc@PNPs at 24h. Scale bar: 10 μm.

Figure 7

Anti-tumor effect in vivo. (A) The therapeutic schedule of ASEc@PNPs for inhibiting tumor growth (n = 5). (B) Images of isolated tumors on day 14. (C) Relative tumor volume variation of mice after different treatments. (D) Weight of the tumor after different treatments. (E) Average body weight of tumor-bearing mice during treatment. The serum (F) and tumor tissues (G) Ag levels of mice at 7 days. Serum levels of AST, ALP, CK (H), CREA-S, and UA-H (I). (J) H&E, TUNEL, and Ki67 staining images of the tumor tissues. Scale bar: 100 μm (**p <0.01, ***p <0.001, ****p < 0.0001).

Figure 8

In vivo CRT and anti-tumor immunity. (A) Immunofluorescence images of tumor sections after CRT staining (pink) assay. Scale bar: 2 mm. (B) IFN- γ, (D)TNF- α, and (C) IL-6 levels in the serum of mice treated as indicated 14 days after injection. (E) Immunofluorescence staining of CD4⁺ T cells (green) and CD8⁺ T cells (red) in the tumor tissues. Scale bar: 100 μm (*p <0.05, **p <0.01, ***p <0.001, ****p < 0.0001).

Figure 9

In vivo anti-pulmonary metastasis effect. (A) Schematic illustration of the schedule for drug treatment. (B) H&E analysis of the lung. Scale bars: 1mm, 200 μm. (C) Immunofluorescence assay of the α-SMA protein (red arrow). Scale bars:100 μm.