Proton pump inhibitors reduce chemotherapeutic hepatotoxicity and enhance hepatic uptake and accumulation of drug-loaded extracellular vesicles

Abstract

Extracellular vesicles (EVs) are involved in the progression of various diseases. Tumor cell-derived EVs (TEVs) are a particular concern, as they can induce fatty liver by promoting liver macrophages to secrete tumor necrosis factor (TNF), thus enhancing the toxicity of chemotherapy. Therefore, reducing pathogenic EV production is a potential strategy for treating EV-related diseases. However, there are currently no effective clinical reagents to obtain this purpose. In addition, EVs are also natural and ideal drug-delivery vehicles. Improving the delivery efficiency of EVs remains a challenge. Proton pump inhibitors (PPIs) have been demonstrated to promote cell uptake of EVs by inducing micropinocytosis. Here, we show that PPIs can accelerate TEV clearance, reduce TEV uptake by liver macrophages and decrease the mRNA expression of TNF in liver macrophages of tumor-bearing mice. Correspondingly, the fatty liver phenotypes are alleviated, and the tolerance to chemotherapy is improved in these mice. Furthermore, our findings indicate that PPIs facilitate the uptake of red blood cell-derived EVs (RBC-EVs) loaded with antisense oligonucleotides of Trim21 (Trim21-ASOs) by the liver macrophages of obesity. Consequently, the inhibition of macrophage inflammatory responses in obese mice mediated by RBC-EVs/Trim21-ASOs was further enhanced by PPIs, resulting in a more profound improvement in obesity and related metabolic disorders. In conclusion, our findings demonstrated that PPIs can effectively clear pathogenic EVs and enhance the delivery efficacy of EV vehicles, making them a highly promising clinical prospect.

Keywords: Chemotherapeutic hepatotoxicity; Delivery efficacy; Extracellular vesicles; Proton pump inhibitors; Rabeprazole.

Fig. 1

Intratumoral injection of PPIs reduce TEVs uptake by liver macrophages. (A) Cre and EV markers in MC38-EVs or Cre⁺ MC38-EVs were detected by western blot. (B) Liver macrophages from Rosa-LSL-tdTomato mice were cocultured with 5 µg Cre⁺ MC38-EVs for 24 h, and the tdTomato expression in isolated F4/80⁺ liver macrophages was detected by flow cytometry. (C–E) Rosa-LSL-tdTomato mice bearing Cre⁺ MC38 tumor were intratumorally injected with DMSO or 15 µg Rabe for 3 days. CD63⁺Cre⁺ EV levels in the blood of these mice were measured by ELISA (C). Representative tdTomato expression in livers of these mice. Scale bar, 50 μm (D). Tnfa mRNA in liver macrophages from these mice was measured by Real-time PCR (E). *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed Student’s t-test; mean ± SD).

Fig. 2

PPIs improve chemotherapeutic tolerance of tumor mice. (A) Representative BODIPY staining of livers from MC38 and Atp6v1a^KD MC38 tumor mice with DMSO or Rabe intratumoral treatment. Scale bar, 50 μm. (B) Real-time PCR analysis of Cyp genes in hepatocytes of MC38 and Atp6v1a^KD MC38 tumor mice with DMSO or Rabe intratumoral treatment. (C) Analysis of RBCs and reticulocytes in MC38 and Atp6v1a^KD MC38 tumor mice treated with dacarbazine alone or combined with Rabe. (D) Analysis of RBCs and reticulocytes in MC38 tumor mice, treated with dacarbazine alone or combined with Ome. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 (unpaired two-tailed Student’s t-test in B; one-way ANOVA followed by Tukey test in C,D; mean ± SD).

Fig. 3

PPIs promote RBC-EVs/Trim21-ASO-mediated inhibition of macrophage inflammatory responses. (A–C) The morphology (A), size distribution (B) and EV markers (C) of the RBC-EVs were evaluated by transmission electron microscopy. Scale bar, 500 nm (A), nanoparticle tracking analysis (B) and western blot (C), respectively. (D) Cy3-conjugated cholesterol-modified Trim21-ASOs were loaded into RBC-EVs and analyzed by flow cytometry. (E) PEMs were treated with 10 µg RBC-EVs/Trim21-ASOs for 48 h. TRIM21 protein expression in PEMs was detected by western blot. (F) PEMs were cocultured with 10 µg RBC-EVs/Trim21-ASOs for 48 h together with or without 10 µM Rabe and then stimulated with 50 µM PA for 12 h. IL-1β, IL-6 and TNF secreted from these cells were measured by ELISA. *P < 0.05; **P < 0.01 (one-way ANOVA followed by Tukey test; mean ± SD).

Fig. 4

PPIs enhance the therapeutic effects of RBC-EVs/Trim21-ASOs on obesity-related metabolic disorders. (A) Mice were intravenously injected with 50 µg PKH26-labeled RBC-EVs/Trim21-ASOs and simultaneously received intraperitoneal injection with 5 mg kg⁻¹ Rabe. RBC-EVs/Trim21-ASOs in isolated F4/80⁺ liver macrophages were analyzed by flow cytometry. (B–F) Male mice were fed an HFD for 16 weeks and then injected with RBC-EVs/Trim21-ASOs alone or combined with Rabe from a specific time point. Body weights (B), GTT (C), ITT (D), plasma ALT and AST levels (E), representative ORO staining of liver sections (F). Scale bar, 50 μm. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey test except for unpaired two-tailed Student’s t-test in A; mean ± SD).