An orally administered bacterial membrane protein nanodrug ameliorates doxorubicin cardiotoxicity through alleviating impaired intestinal barrier

Abstract

The cardiotoxicity caused by Dox chemotherapy represents a significant limitation to its clinical application and is a major cause of late death in patients undergoing chemotherapy. Currently, there are no effective treatments available. Our analysis of 295 clinical samples from 132 chemotherapy patients and 163 individuals undergoing physical examination revealed a strong positive correlation between intestinal barrier injury and the development of cardiotoxicity in chemotherapy patients. We developed a novel orally available and intestinal targeting protein nanodrug by assembling membrane protein Amuc_1100 (obtained from intestinal bacteria Akkermansia muciniphila), fluorinated polyetherimide, and hyaluronic acid. The protein nanodrug demonstrated favorable stability against hydrolysis compared with free Amuc_1100. The in vivo results demonstrated that the protein nanodrug can alleviate Dox-induced cardiac toxicity by improving gut microbiota, increasing the proportion of short-chain fatty acid-producing bacteria from the Lachnospiraceae family, and further enhancing the levels of butyrate and pentanoic acids, ultimately regulating the homeostasis repair of lymphocytes in the spleen and heart. Therefore, we believe that the integrity of the intestinal barrier plays an important role in the development of chemotherapy-induced cardiotoxicity. Protective interventions targeting the intestinal barrier may hold promise as a general clinical treatment regimen for reducing Dox-induced cardiotoxicity.

Keywords: Doxorubicin cardiotoxicity; Homeostasis of lymphocytes; Intestinal barrier; Oral nanodrugs; Protein delivery.

Graphical abstract

Scheme 1

Diagram depicting the synthesis of the membrane protein drug HFPA and its mechanism in mediating.

Fig. 1

The analysis of intestinal barrier permeability and cardiotoxicity in chemotherapy patients. (A)The diagram of clinical sample screening process; Plasma NT-proBNP (B), cTnT (C), LPS (D), and zonulin family peptide (E) levels in chemotherapy patients were significantly higher than those in healthy controls. Simple linear regression analysis showed that the levels of LPS (F and H) and zonulin (G and I) were positively correlated with plasma NT-proBNP and cTnT levels. Using t-test to analyze the statistical differences. * P-value <0.05, ** P-value <0.01.

Fig. 2

Dox-induced intestinal barrier injury precedes the onset of abnormal heart function. (A) H&E staining showed the dynamic process of ileal morphology injury induced by Dox during 6–240 h. Scale bar: 100 μm; Dynamic changes in serum LPS (B) and zonulin levels and (C); (D–E) Immunofluorescence images of ileal sections: nuclei, DAPI (blue); ZO-1 (red), Scale bar: 50 μm. (F–H) Plasma levels of the cardiac injury markers NT-proBNP, cTnT, and CKMB; (I–K) Echocardiographic analysis of cardiac function in mice treated by Dox and PBS during 6–240 h, respectively. EF, ejection fraction; FS, fractional shortening. n = 6 mice/group, mean ± SD, and statistical significance was analyzed using the two-way ANOVA followed by Tukey's multiple comparison. * P-value <0.05, ** P-value <0.01.

Fig. 3

Early intestinal barrier injury can aggravate Dox cardiotoxicity. (A) Schematic protocol for mice treatments. 2.5% DSS was administered in sterile drinking water every 5 days followed by 7 days of DSS-free water for 3 cycles to induce chronic enteritis model; (B–D) Estimation on cardiac function of mice treated by PBS, Dox, DSS and DSS + Dox echocardiography; (E–G) Plasma levels of the cardiac injury markers NT-proBNP, cTnT, and CKMB; (H–I) Plasma levels of LPS and zonulin; (J) Estimation on ileal histopathology morphology of mice based on H&E staining. Scale bar: 100 μm; EF, ejection fraction; FS, fractional shortening. n = 6 mice/group, mean ± SD, and statistical significance was analyzed using the two-way ANOVA. * P-value <0.05, ** P-value <0.01.

Fig. 4

Characterization of HFPA. (A) Procedure for HFPA synthesis. (B) TEM image of HFPA. (C) Size of HFPA. (D) Zeta potential of HFPA and FPA. (E) FTIR of HFPA and FPA. (F) SDS-PAGE analysis from Amuc_1100 and HFPA treated by trypsin (0–180 min). (G) HFPA in mice and intestinal tract at different time.

Fig. 5

HFPA enhances intestinal barrier integrity and alleviates cardiac dysfunction, myocardial fibrosis and apoptosis in Dox-treated mice. (A) Schematic protocol for mice treatments. Mice were injected intravenously with a cumulative dose of 15 mg/kg Dox three times in 21 days to construct chronic Dox cardiotoxicity model. The mice were respectively treated twice a week with HFPA (3 μg/200 μL) by oral gavage for 7 days before the first Dox treatment; (B) Estimation on ileal histopathology morphology of Dox mice after HFPA intervention based on H&E staining. Scale bar: 100 μm; (C–D) Plasma levels of LPS and zonulin; (E–F) Immunofluorescence images of ileal sections: DAPI (blue); ZO-1 (red). Scale bar: 50 μm; (G–I) Echocardiographic analysis the differences of cardiac function in mice treated by HFPA and the other groups. EF, ejection fraction; FS, fractional shortening; (J–L) Plasma levels of the cardiac injury markers NT-proBNP, cTnT, and CKMB; (M − O) Myocardial fibrosis and cardiomyocyte apoptosis was evaluated by Masson staining and TUNEL staining. n = 6 mice/group, mean ± SD, and statistically analyzed using one-way ANOVA followed by Tukey's multiple comparison. * P-value <0.05, ** P-value <0.01.

Fig. 6

Oral HFPA administration attenuated Dox-induced gut microbial dysbiosis and inflammation. (A) PCoA was conducted to visualize differences in the fecal microbiota structure among the four groups; (B) α-diversity (Chao1 index) of the intestinal microbiota; (C–D) Relative abundance of intestinal microbiota constituents at the phylum and genus levels; (E–F) Species with significant differences in relative abundance at the genus level; (G) Analysis of the differences in the intestinal microbiota by LEfSe (LDA score≥3，P-value <0.05); (H–I) Contents of butyric acid and pentanoic acid in feces of mice; (J–M) Inflammatory cytokines IL-1β, IFN-γ, TNF-α and MCP-1 levels in plasma, respectively. Values are expressed as mean ± SD; n = 4–6 mice/group. Statistical significance was analyzed using the one-way ANOVA followed by Tukey's multiple comparison. * P-value <0.05, ** P-value <0.01.

Fig. 7

HFPA alleviates Dox cardiotoxicity by remodeling the composition of cardiac immune cells. (A–D) Proportion of lymphocyte (Lymphocyte, B, Th, Treg cells) and (E–F) myeloid derived immune cells (Macrophage and Neutrophil) in the heart. n = 4 mice/group, mean ± SD. Statistically analyzed using one-way ANOVA followed by Tukey's multiple comparison. * P-value <0.05, ** P-value <0.01.

Fig. 8

Protecting the intestinal barrier to alleviate Dox cardiotoxicity may be a general treatment strategy. (A) Schematic protocol for mice treatments. Construction of chronic Dox cardiotoxicity mouse model. The mice were respectively treated every two days with LA (0.4 mg/200 μL) by oral gavage, or GLP-2 (0.5 μg/200 μL) was administrated via intraperitoneal injections; (B–D) Echocardiography showed that LA and GLP-2 also prevented Dox induced left ventricular dysfunction, n = 6 mice/group; (E–G) Plasma NT-proBNP, cTnT, and CKMB levels in Dox mice treated with LA and GLP-2, n = 6 mice/group; (H) LA and GLP-2 significantly decreased plasma LPS levels, n = 6 mice/group; (I, L) Myocardial fibrosis and cardiomyocyte apoptosis were evaluated by Masson staining and (J, M) TUNEL staining; (K, N) Immunofluorescence images of ileal sections: DAPI (blue); ZO-1 (red). Scale bar: 50 μm; n = 6 mice/group. Mean ± SD, and statistically analyzed using two-way ANOVA followed by Tukey's multiple comparison. * P-value <0.05, ** P-value <0.01.