Tea leaf-derived exosome-like nanotherapeutics retard breast tumor growth by pro-apoptosis and microbiota modulation

Abstract

While several artificial nanodrugs have been approved for clinical treatment of breast tumor, their long-term applications are restricted by unsatisfactory therapeutic outcomes, side reactions and high costs. Conversely, edible plant-derived natural nanotherapeutics (NTs) are source-widespread and cost-effective, which have been shown remarkably effective in disease treatment. Herein, we extracted and purified exosome-like NTs from tea leaves (TLNTs), which had an average diameter of 166.9 nm and a negative-charged surface of - 28.8 mV. These TLNTs contained an adequate slew of functional components such as lipids, proteins and pharmacologically active molecules. In vitro studies indicated that TLNTs were effectively internalized by breast tumor cells (4T1 cells) and caused a 2.5-fold increase in the amount of intracellular reactive oxygen species (ROS) after incubation for 8 h. The high levels of ROS triggered mitochondrial damages and arrested cell cycles, resulting in the apoptosis of tumor cells. The mouse experiments revealed that TLNTs achieved good therapeutic effects against breast tumors regardless of intravenous injection and oral administration through direct pro-apoptosis and microbiota modulation. Strikingly, the intravenous injection of TLNTs, not oral administration, yielded obvious hepatorenal toxicity and immune activation. These findings collectively demonstrate that TLNTs can be developed as a promising oral therapeutic platform for the treatment of breast cancer.

Keywords: Apoptosis; Breast cancer; Intestinal microbiota rebalance; Natural nanomedicine; Reactive oxygen species.

Scheme 1.

Schematic diagram of the extraction and purification processes of TLNTs and their application in the treatment of breast cancer via i.v. injection and oral administration. A The production processes of TLNTs based on differential centrifugation accompanied by density gradient centrifugation. B The in vivo anti-tumor mechanism and comparative therapeutic outcomes of TLNTs against breast cancer via i.v. injection and oral administration

Fig. 1

Physicochemical and functional characterizations of TLNTs. A TLNTs in the sucrose gradients after ultracentrifugation. B TEM imaging (scale bar: 100 nm), C AFM imaging, D hydrodynamic particle size distribution, E lipid compositions, F protein summary, G KEGG annotated statistical charts and H Go secondary classification statistical charts of TLNTs. I Flavonoids and J polyphenols in TLNTs. EGCG epigallocatechin gallate, ECG epicatechin gallate

Fig. 2

In vitro anti-tumor effects of TLNTs. A Cytotoxicity of TLNTs against various tumor cell lines after co-incubation with TLNTs at protein concentrations from 0.5 to 64 µg/mL for 24 and 48 h, respectively. Each point represents the mean ± S.E.M. (n = 5). B Pro-apoptotic properties of TLNTs after co-incubation with TLNTs for 4 and 8 h, respectively. C CLSM images of 4T1 cells stained with DCFH-DA after co-incubation with TLNTs for 4 and 8 h, respectively (scale bar: 50 μm). D ROS fluorescence intensity of 4T1 cells after co-incubation with TLNTs for 4 and 8 h, respectively. E Mitochondrial membrane potential changes in 4T1 cells (scale bar: 50 μm). F TLNTs restrained cell cycle progression in 4T1 cells after co-incubation with TLNTs for 12 and 24 h, respectively. Populations of 4T1 cells in various cell cycle phases were determined by FCM. Each point represents the mean ± S.E.M. (n = 3; *p < 0.05 and **p < 0.01). G Western blot analysis of 4T1 cells receiving the treatment of TLNTs for 48 h. Cyclin A, cyclin B and cyclin D proteins were probed. GAPDH was probed to ensure the equal loading of total proteins in each lane

Fig. 3

In vivo bio-distribution profiles of TLNTs. A Fluorescence images of tumors, five major organs (heart, liver, spleen, lung and kidney) and the GIT from breast tumor-bearing mice receiving the treatment of DiR-TLNTs via i.v. injection and oral administration at different time points (6, 12, 24 and 48 h). B Distribution profiles of TLNTs in different sections of the GIT following oral administration of DiO-labeled TLNTs for 6 h (scale bar: 100 µm)

Fig. 4

In vivo anti-breast tumor effects of TLNTs. A Body weight variations and B tumor growth curves of different mouse groups with various treatments during the whole experimental period. C Tumor weights, D representative tumor images and E spleen weights of different mouse groups with various treatments at the end of the experiment. Each point represents the mean ± S.E.M. (n = 5; *p < 0.05, **p < 0.01, ***p < 0.001 and ns no significance). F H&E- and TUNEL-stained tumor sections showing pathological changes and apoptosis profiles (scale bar: 100 μm)

Fig. 5

Transcriptome analysis of tumors from various mouse groups. A Volcano plot of differentially expressed genes (DEGs) between the control group and the LTNT (i.v., high)-treated group. B Volcano plot of DEGs between the control group and the LTNT (oral, high)-treated group. C Venn diagram showing overlapped genes among the control group, the LTNT (i.v., high)-treated group and the LTNT (oral, high)-treated group. D Heatmap showing significantly up-regulated and down-regulated genes in the tumors from mouse groups receiving various treatments (fold change ≥ 2 and p < 0.05). E Gene Ontology (GO) enrichment analysis for genes in the brown module. The color represents the adjusted p-values, and the sizes of the spots represent the gene numbers. (F) KEGG pathway analysis for cell apoptosis-associated genes (n = 3)

Fig. 6

Evaluation of remodeling effects of TLNTs on the intestinal microbiota. A α-Diversities were presented by box plots of the Simpson indexes. B Principal coordinates analysis (PCoA) of the intestinal microbiota. C Venn diagram of common and unique bacterial species of mice in each group. (D) Total numbers of microbial species in each group at the OUT level. E Relative abundance of intestinal microbiome. Genus-level taxonomy is presented as the percentage of total sequences. F Microbial compositions of various mouse groups at the phylum level. G–J Relative abundance of beneficial bacteria and harmful bacteria in each group. Each point represents the mean ± S.E.M. (n = 3; *p < 0.05)

Fig. 7

In vivo biosafety evaluation of TLNTs after i.v. injection and oral administration. A Body weight variations, B organ indices, C pro-inflammatory cytokine levels of various mouse groups. The concentrations of D Complement C3, E ALT, F AST, G BUN and H CRE in plasma from mice receiving the treatment of TLNTs via i.v. and oral routes. Each point represents the mean ± S.E.M. (n = 3; *p < 0.05, **p < 0.01 and ***p < 0.001)