Tumor microenvironment responsive nano-herb and CRISPR delivery system for synergistic chemotherapy and immunotherapy

Abstract

Chemoresistance remains a significant challenge for effective breast cancer treatment which leads to cancer recurrence. CRISPR-directed gene editing becomes a powerful tool to reduce chemoresistance by reprogramming the tumor microenvironment. Previous research has revealed that Chinese herbal extracts have significant potential to overcome tumor chemoresistance. However, the therapeutic efficacy is often limited due to their poor tumor targeting and in vivo durability. Here we have developed a tumor microenvironment responsive nanoplatform (H-MnO₂(ISL + DOX)-PTPN2@HA, M(I + D)PH) for nano-herb and CRISPR codelivery to reduce chemoresistance. Synergistic tumor inhibitory effects were achieved by the treatment of isoliquiritigenin (ISL) with doxorubicin (DOX), which were enhanced by CRISPR-based gene editing to target protein tyrosine phosphatase non-receptor type 2 (PTPN2) to initiate long-term immunotherapy. Efficient PTPN2 depletion was observed after treatment with M(I + D)PH nanoparticles, which resulted in the recruitment of intratumoral infiltrating lymphocytes and an increase of proinflammatory cytokines in the tumor tissue. Overall, our nanoparticle platform provides a diverse technique for accomplishing synergistic chemotherapy and immunotherapy, which offers an effective treatment alternative for malignant neoplasms.

Keywords: CRISPR-Cas 9; Doxorubicin (DOX); Immunotherapy; Isoliquiritigenin (ISL); Protein tyrosine phosphatase non-receptor type 2 (PTPN2); Synergistic therapy.

Scheme 1

Design of M(I + D)PH nanocomposite and its antitumor therapeutic function. Schematic diagram of the preparation routes of M(I + D)PH nanoparticle and the synergistic chemotherapy and immunotherapy in vivo

Fig. 1

Synergistic inhibitory effects of DOX and ISL on cell viability of 4T1 cells. A–B Chemical formula of DOX (A) and ISL (B). C Cell viability of 4T1 cells treatment of different concentrations of DOX (n = 3). D Cell viability of 4T1 cells treatment of different concentrations of ISL (n = 3). E 4T1 cells treated with DOX of different concentrations in combination of ISL (n = 3). F The synergistic score of DOX combined with ISL calculated by using the Synergyfinder 3.0 software. *: P < 0.05. **: P < 0.01. ***: P < 0.001

Fig. 2

Characterization of M(I + D)PH nanoparticles. A TEM images of SiO₂, SiO₂@PDA@MnO₂ and H-MnO₂ nanoparticles. Scale bar: 50 nm B Representative photos of SiO₂, SiO₂@PDA@MnO₂, H-MnO₂. C–D The linear relationship between the UV–vis absorbance and concentration of DOX C and ISL D (DOX: 485 nm, ISL: 365 nm). E The loading weight ratios of drugs in H-MnO₂ nanoparticles obtained at different feeding drug: H-MnO₂ ratios. F Drugs release profiles from M(I + D) nanoparticles in different conditions within 24 h (n = 3). G The UV–vis spectra of DOX, ISL and M(I + D). H Agarose gel electrophoresis of M/PAH nanoparticles loading plasmids compared to the control (0.5 µg). I FTIR spectra of HA, M and MH nanoparticles. J The zeta potential of M, M(I + D), M(I + D)/PAH, M(I + D)P and M(I + D)PH nanoparticles (n = 3)

Fig. 3

Cell viability and gene editing mediated by M(I + D)PH in vitro. A Cellular uptake of M(I + D)P and M(I + D)PH nanoparticles. Scale bar: 20 µm. B Evaluation of PTPN2 gene editing efficiency in 4T1 cells with indicated treatments. C Protein expression level was detected by western blotting. D Representative images for flow cytometry of 4T1 cells with different treatments. I: PBS, II: MH, III: I + D, IV: M(I + D)H, V: MPH, VI: M(I + D)PH. E Sphere-forming capacities of 4T1 cells cultured with different treatments. Quantification of sphere number was shown. ***: P < 0.001. I: PBS, II: MH, III: I + D, IV: M(I + D)H, V: MPH,VI: M(I + D)PH. F The representative fluorescent images of tumor cells by treatment of different treatments dyed with Calcein-AM/PI. Green, Calcein-AM; Red, PI. Scale bar: 50 µm

Fig. 4

In vivo antitumor efficacy. A Schematic illustration of the administration design. B Body weight changes of mice after various treatments. C Tumor growth curves of 4T1 tumor-bearing mice after intravenous administrations. D Representative photos of the sacrificed tumor with different treatments. E Tumor weights of the mice. F Western blots analysis of PTPN2 and GAPDH expression of the tumor. G T7EI assay of tumor tissue after various treatments. H–I Representative ex vivo fluorescence image of the major organs and tumor H from an 4T1 cells tumor-bearing mouse at 10 h after M(I + D)PH injection and mean fluorescence intensity of major organs and tumor (I). J Representative images of H&E staining, IHC for Ki67 and PTPN2, and apoptosis in tumor sections. I: PBS, II: MH, III: I + D, IV: M(I + D)H, V: MPH, VI: M(I + D)PH. ***: P < 0.001

Fig. 5

Antitumor immune effects and the related mechanisms. A–C Representative flow cytometry plots and the quantitative analysis of CD3⁺ and CD8.⁺T cells (A) Matured DCs (B) and Treg cells (C) in tumor tissues after different treatments. D–F The cytokines levels of IL-6 (D), IFN-γ (E), and TNF-α (F) in tumor tissues after different treatments. I: PBS, II: MH, III: I + D, IV: M(I + D)H, V: MPH, VI: M(I+ D)PH. *: P < 0.05. **: P < 0.01. ***: P < 0.001