Tumor cell membrane-camouflaged responsive nanoparticles enable MRI-guided immuno-chemodynamic therapy of orthotopic osteosarcoma

Abstract

Osteosarcoma is a refractory bone disease in young people that needs the updating and development of effective treatment. Although nanotechnology is widely applied in cancer therapy, poor targeting and inadequate efficiency hinder its development. In this study, we prepared alendronate (ALD)/K7M2 cell membranes-coated hollow manganese dioxide (HMnO₂) nanoparticles as a nanocarrier to load Ginsenoside Rh2 (Rh2) for Magnetic Resonance imaging (MRI)-guided immuno-chemodynamic combination osteosarcoma therapy. Subsequently, the ALD and K7M2 cell membranes were successively modified on the surface of HMnO₂ and loaded with Rh2. The tumor microenvironment (TME)-activated Rh2@HMnO₂-AM nanoparticles have good bone tumor-targeting and tumor-homing capabilities, excellent GSH-sensitive drug release profile and MRI capability, and attractive immuno-chemodynamic combined therapeutic efficiency. The Rh2@HMnO₂-AM nanoparticles can effectively trigger immunogenic cell death (ICD), activate CD4⁺/CD8⁺ T cells in vivo, and upregulate BAX, BCL-2 and Caspase-3 in cellular level. Further results revealed that Rh2@HMnO₂-AM enhanced the secretion of IL-6, IFN-γ and TNF-α in serum and inhibited the generation of FOXP3⁺ T cells (Tregs) in tumors. Moreover, the Rh2@HMnO₂-AM treatment significant restricted tumor growth in-situ tumor-bearing mice. Therefore, Rh2@HMnO₂-AM may serve as an effective and bio-friendly nanoparticle platform combined with immunotherapy and chemodynamic therapy to provide a novel approach to osteosarcoma therapy.

Keywords: Chemodynamic therapy; Ginsenoside Rh2; Hollow manganese dioxide; Immunotherapy; Magnetic resonance imaging.

Graphical abstract

Scheme 1

Synthetic procedure of Rh2@HMnO₂-AM and mechanism of MRI-guided immuno-chemodynamic synergistic osteosarcoma therapy.

Fig. 1

(A) TEM images of (a) sSiO₂, (b) sSiO₂-MnO₂, (c) HMnO₂ and (d) HMnO₂-AM. (B) Elemental mapping of HMnO₂-AM nanoparticles, including bright field, Mn, O, Na, P and N. (C) FTIR spectra of HMnO₂, cell membrane and HMnO₂-AM. (D) XRD pattern of HMnO₂ and HMnO₂-AM. (E) Zeta potential of sSiO₂, sSiO₂-MnO₂, HMnO₂ and HMnO₂-AM. (F) The stability of Rh2@HMnO₂-AM in different solutions (FBS, H₂O and DMEM medium).

Fig. 2

(A) Cytotoxicity evaluation of HUVEC, K7M2 cell and RAW 264.7 with different concentrations of HMnO₂-AM nanoparticles for 24 h. (B) Hemolysis ratio and inset image of RBCs treated with various concentrations of HMnO₂-AM nanoparticles. (C) UV–vis absorption spectra and images (inset) of MB degradation via Mn²⁺-triggered Fenton-like reaction with or without GSH (0, 1, 10 mM). (D) MB degradation via the Mn²⁺-triggered Fenton-like process in different solutions with different solvent (H₂O or NaHCO₃/CO₂). (E) MB degradation by H₂O₂ plus GSH-treated Rh2@HMnO₂-AM nanoparticles. (F) MB degradation by 2 mM GSH plus different concentration of Rh2@HMnO₂-AM. UV–vis absorption spectra of HMnO₂-AM nanoparticles in (G) pH 6.5 for 1 h, 4 h, 8 h, and 12 h. (H) UV–vis absorption spectra of HMnO₂-AM nanoparticles in different concentration (0, 0.5, 2 and 10 mM) of GSH for 3 min at pH 7.4. (I) Drug releasing of Rh2@HMnO₂-AM nanoparticles in pH 7.4, pH 6.5, pH 7.4 + 2 mM GSH and pH 6.5 + 2 mM GSH.

Fig. 3

(A) Scheme of K7M2 tumor cell co-culture system. K7M2 tumor cells were seeded in the upper chamber while DCs were cultured in lower chamber. (B) Western blot analysis of HMGB1 expression on K7M2 cells after different treatments. (C) Flow cytometric analysis and (D) quantification of DCs maturation after 24 h co-culture with K7M2 cells through treating with PBS, Rh2, HMnO₂-AM, Rh2@HMnO₂ or Rh2@HMnO₂-AM. (E) Western blot analysis and (F) quantification of BAX, BCL-2 and Caspase 3 of different groups.

Fig. 4

(A) The CLSM images of K7M2 cells or RAW264.7 cells treated by DOX@HMnO₂ and DOX@HMnO₂-AM nanoparticles for 3 h (scale bar = 20 μm). (B) The Calcian-AM/PI staining of K7M2 cells after various treatments of PBS, HMnO₂-AM, Rh2, HMnO₂-AM + GSH and Rh2@HMnO₂ + GSH. The green fluorescence shows living cells and the red fluorescence represents dead cells. (C) DCFH-DA fluorescence of K7M2 cells exposed to MnCl₂ and Rh2@HMnO₂-AM with 2 mM GSH.

Fig. 5

In vivo imaging of Rh2@HMnO₂-AM nanoparticles in K7M2 tumor-bearing mice. (A) Fluorescence imaging of K7M2 tumor-bearing mice at pre-injection at different time points after injection of control, Cy5.5 and Cy5.5@HMnO₂-AM groups. (B) The statistical fluorescence intensity of tumors in different groups. (C) The image of K7M2-bearing mice (the red circle marks tumor site). (D) Ex vivo fluorescence imaging of main organs and tumor in Rh2@HMnO₂-AM group. (E) T1-weighted MR images of the Rh2@HMnO₂-AM. (F) T1-MR images of K7M2 tumor-bearing mice after injection of Rh2@HMnO₂-AM at different time points.

Fig. 6

(A) Tumor photos of mice in 15 days of different groups. (B) The body weight of K7M2 tumor-bearing mice at different time points. (C) Relative tumor volume of mice after various treatments. (D) Survival curves of K7M2-tumor bearing mice after different treatments. (E) Immunohistochemical analysis of HMGB-1 and CRT, and immunofluorescence staining of Tregs in tumor tissue. (F) Representative immunofluorescence staining of CD4⁺ (red) and CD8⁺ (green) T cells in spleen. Scale bar represents 100 μm for each panel. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

(A) Representative immunofluorescence staining of CD3⁺ and CD8⁺ T cells in tumor tissue. (B) Flow cytometric examination and (C) quantitative analysis of intratumor infiltration of CD3⁺ and CD8⁺ T cells. Contents of the (D) IL-6, (E) TNF-α and (F) IFN-γ in serum of mice on the 14th day after treatment with PBS, Rh2, HMnO₂-AM, Rh2@HMnO₂ or Rh2@HMnO₂-AM. Scale bar represents 100 μm for each panel.