Nanofactory for metabolic and chemodynamic therapy: pro-tumor lactate trapping and anti-tumor ROS transition

Abstract

Lactate plays a critical role in tumorigenesis, invasion and metastasis. Exhausting lactate in tumors holds great promise for the reversal of the immunosuppressive tumor microenvironment (TME). Herein, we report on a "lactate treatment plant" (i.e., nanofactory) that can dynamically trap pro-tumor lactate and in situ transformation into anti-tumor cytotoxic reactive oxygen species (ROS) for a synergistic chemodynamic and metabolic therapy. To this end, lactate oxidase (LOX) was nano-packaged by cationic polyethyleneimine (PEI), assisted by a necessary amount of copper ions (PLNP^Cu). As a reservoir of LOX, the tailored system can actively trap lactate through the cationic PEI component to promote lactate degradation by two-fold efficiency. More importantly, the byproducts of lactate degradation, hydrogen peroxide (H₂O₂), can be transformed into anti-tumor ROS catalyzing by copper ions, mediating an immunogenic cell death (ICD). With the remission of immunosuppressive TME, ICD process effectively initiated the positive immune response in 4T1 tumor model (88% tumor inhibition). This work provides a novel strategy that rationally integrates metabolic therapy and chemodynamic therapy (CDT) for combating tumors.

Keywords: Chemodynamic therapy; Immunogenic cell death; Immunosuppressive tumor microenvironment; Lactate.

Scheme 1

Schematic illustration of the lactate exhaustion process of PLNP^Cu nanosystem through extracellular lactate active-adsorption and establishment of the intracellular lactate treatment plant

Fig. 1

Characterization of PLNP^Cu. a, b TEM images of PLNP^Cu at pH 7.4. c TEM images of PLNP^Cu at pH 6.5. d DLS of PLNP^Cu at pH 7.4. e The pH-triggered charge rebound behavior of PLNP^Cu (n = 3). f Maintenance of size stability of PLNP^Cu in PBS and PBS with 5% FBS (at pH 7.4) (n = 3). g LOX loading capacities on LNP and LNP^Cu respectively (n = 3). h Cumulative release of LOX from PLNP^Cu in PBS at pH 7.4 and pH 6.5 (n = 3). i Hemolysis rate of PLNP^Cu at different PEI concentrations (n = 3). Results were expressed as mean ± SD. The significant difference was calculated via two-tailed t-test analysis (g). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Scheme 2

Schematic illustration of the PLNP^Cu nanofactory closed-loop for lactate consumption and conversion

Fig. 2

Functions verification of lactate treatment plant PLNP^Cu in vitro. a Lactate adsorption rate of PEI in nanoparticles (n = 3). b Lactate depletion effect of LNP^Cu overtime at pH 7.4 (n = 3). c Lactate degradation ratio of LNP^Cu at the shorter incubation time in different acidic solutions (n = 3). d Lactate degradation rate of LNP^Cu compared with LOX after sufficiently enzymatic hydrolysis (n = 3). e The generation of enzymatic product H₂O₂ (n = 3). f The degradation process of MB caused by ·OH generation from the Fenton-like reaction of nanoparticles under different conditions. Results were expressed as mean ± SD. The significant difference was calculated via one-way ANOVA analysis (a, d, e). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Intracellular behaviors of PLNP^Cu. a The cytotoxicity of PLNP^Cu and LOX against 4T1 cells (n = 5). b The flow cytometry analysis of cellular uptake of different agents (LOX@FITC, LNP^Cu@FITC, PLNP^Cu@FITC pH = 7.4 and PLNP^Cu@FITC pH = 6.5) after 3 h of incubation with 4T1 cells. (n = 3) c The lactate consumption analysis in 4T1 cell supernatant after treatment with PLNP^Cu. (n = 3) d Fluorescent microscopy image of intracellular ROS generation. Scale bars, 100 μm. e, f The flow cytometry analysis of ROS generation after treatment with PBS, LOX, PLNP^Cu pH = 6.5 and PLNP^Cu pH = 7.4. Results were expressed as mean ± SD. The significant difference was calculated via one-way ANOVA analysis (b, c, f). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 4

ICD induction effects of PLNP^Cu in vitro. a, b Flow cytometric analysis of apoptosis rate of 4T1 cells after treatment with PBS, LOX, PLNP^Cu pH = 7.4 and PLNP^Cu pH = 6.5. c, d The flow cytometric analysis and quantitative results of relative fluorescence intensity of CRT on 4T1 cells. e Fluorescent microscopy image of CRT exposure. Scale bars, 100 μm. f Fluorescent microscopy image of HMGB1 outflow from the nucleus. Scale bars, 100 μm. g The ATP secretion from cancer cells after different treatments. Results were expressed as mean ± SD. The significant difference was calculated via one-way ANOVA analysis (b, d, g). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5

Immunomodulatory effects on macrophages of PLNP^Cu in vitro. a, c The flow cytometric analysis and quantitative results of CD80 (M1 macrophages marker) on RAW 264.7 macrophages after incubation with PLNP^Cu for different times (3, 6, 24 h). b, d The flow cytometric analysis and quantitative results of CD206 (M2 macrophages marker) on RAW 264.7 macrophages after treatment. e Immunofluorescence examination of RAW 264.7 macrophages after incubation with PLNP^Cu for 24 h. Scale bars, 50 μm. f, gThe flow cytometric analysis and quantitative results of CD80 and CD206 on M2-macrophages after treatment. Results were expressed as mean ± SD. The significant difference was calculated via one-way ANOVA analysis (c, d, g). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 6

Antitumor effects of PLNP^Cu in vivo. a The treatment scheme of PLNP^Cu. b, c Individual tumor growth curves and average tumor growth curves of the mice with different treatments. d Body weight of the mice during the therapy. e The lactate consumption effect after treatment in vivo. Results were expressed as mean ± SD. The significant difference was calculated via one-way ANOVA analysis (c, e). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 7

Immunogenic CDT induced an immune-active TME in vivo. a Immunofluorescence staining of intratumor infiltrating CD8⁺ T cells after treatments with PBS, LOX, PLNP and PLNP^Cu. Scale bars, 100 μm. b The flow cytometry analysis of tumor infiltration of CD3⁺CD8⁺ T cells. c, d The flow cytometry analysis of ROS generation after treatments. e Immunofluorescence staining of intratumor ROS generation. Scale bars, 100 μm. Results were expressed as mean ± SD. The significant difference was calculated via one-way ANOVA analysis (b, c). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 8

Immune activation by PLNP^Cu in vivo. a The flow cytometric images of the CD4⁺ T cells and CD8⁺ T cells in spleen (gated on CD3⁺ T cells). b The flow cytometric images of the CD80⁺CD86⁺ DCs in LNs (gated on CD11⁺ DCs). c The flow cytometric images of the CD86⁺MHC II ⁺ DCs in LNs (gated on CD11⁺ DCs). d The flow cytometric quantification of the rate of CD8⁺/CD4⁺ T cells in spleen. e The flow cytometric quantification of the CD86⁺CD80⁺ DCs in LNs. f The CD86⁺MHC II⁺ DCs in LNs. Results were expressed as mean ± SD. The significant difference was calculated via one-way ANOVA analysis (d–f). (NS represented not significant, *p < 0.05, **p < 0.01, ***p < 0.001).