Dual functionalized brain-targeting nanoinhibitors restrain temozolomide-resistant glioma via attenuating EGFR and MET signaling pathways

Abstract

Activation of receptor tyrosine kinase (RTK) protein is frequently observed in malignant progression of gliomas. In this study, the crosstalk activation of epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) signaling pathways is demonstrated to contribute to temozolomide (TMZ) resistance, resulting in an unfavorable prognosis for patients with glioblastoma. To simultaneously mitigate EGFR and MET activation, a dual functionalized brain-targeting nanoinhibitor, BIP-MPC-NP, is developed by conjugating Inherbin3 and cMBP on the surface of NHS-PEG₈-Mal modified MPC-nanoparticles. In the presence of BIP-MPC-NP, DNA damage repair is attenuated and TMZ sensitivity is enhanced via the down-regulation of E2F1 mediated by TTP in TMZ resistant glioma. In vivo magnetic resonance imaging (MRI) shows a significant repression in tumor growth and a prolonged survival of mice after injection of the BIP-MPC-NP and TMZ. These results demonstrate the promise of this nanoinhibitor as a feasible strategy overcoming TMZ resistance in glioma.

Fig. 1. The synthesis and characteristics of nanoinhibitors.

a Schematic illustration and delivery process of the nanoinhibitor, BIP-MPC-NP, enhancing TMZ sensitivity against glioma via intravenous injection. b The schematic diagram revealing the establishment and structure of nanoinhibitors. c The transmission electron microscope (TEM) images of the nanoinhibitors. Scale bar = 50 nm. d The dynamic light scattering (DLS) measurements of BIP-NP and BIP-MPC-NP. e Fluorescence images presenting the co-localization of FITC-MBP and RhB-EBP for each BIP-MPC-nanoparticle. Scale bar = 15 μm. f Förster resonance energy transfer (FRET) analysis of BIP-MPC-NP and MBP/EBP/MPC-NP. g The zeta potential data of BIP-NP and BIP-MPC-NP (n = 3). h The quantitative analysis of peptide fragments per nanoinhibitor (n = 5). i The ratio of EBP and MBP on the nanoparticles (n = 5). j TEM micrograph exhibited the oxygen element (O), nitrogen element (N) and phosphorus element (P). Scale bar = 25 nm. The error bars in g, h and i represent the S.D. of three or five measurements.

Fig. 2. The biological activity of nanoinhibitors.

a The fluorescence assays showed the localization of EBP-MPC-NP, MBP-MPC-NP or BIP-MPC-NP on the LN229R cell surface. The gray value was displayed in line plots. Scale bar = 50 μm. b The flow cytometric analysis of cells with MPC-NP, EBP-MPC-NP, MBP-MPC-NP or BIP-MPC-NP in vitro (n = 3). c The transportation assays showed the permeability of EBP-MPC-NP, MBP-MPC-NP and BIP-MPC-NP in vitro (n = 3). d The binding affinity of MPC-NP, EBP-MPC-NP, MBP-MPC-NP or BIP-MPC-NP after penetrating BBB models. Scale bar = 25 μm. e The IF assays showed the attenuation of p-EGFR and p-MET by administration of MPC-NP, EBP-MPC-NP, MBP-MPC-NP or BIP-MPC-NP in BBB model. Scale bar = 25 μm. f Ex vivo fluorescence and bioluminescence images of the visceral organs after the injection of BIP-NP or BIP-MPC-NP in mice. g, h The histogram summarized the relative luminescence intensity and the relative fluorescence intensity of tumor-bearing brains. The error bars in b, c, g and h represent the S.D. of three measurements (n = 3). P value is determined by Student’s t-test. Significant results are presented as NS non-significant, **P < 0.01, or ***P < 0.001.

Fig. 3. BIP-MPC-NP inhibits DNA damage repair via the mitigation of EGFR and MET signaling pathways.

a The EGFR, p-EGFR, MET and p-MET expression in TMZ-resistant glioma cells treated with different nanoinhibitors. b The levels of EGFR and MET dimers in TMZ-resistant glioma cells treated with BIP-MPC-NP. c The heatmap displayed the differential expressed proteins between GBM patients with low EGFR and MET copy numbers group and with high EGFR and MET copy numbers group. The Sankey diagram displayed the Gene Ontology of the differentially expressed proteins. d The inhibition of DNA damage repair modules expression and the increase of γH2AX expression with the treatments of TMZ and different nanoinhibitors in LN229R, U87MGR and HG9R. e The boxplots showing the statistics of comet assay of TMZ-resistant glioma cells with TMZ and different nanoinhibitors (n = 50). In the box plots, bounds of the box spans from 25 to 75% percentile, center line represents median, and whiskers visualize minimum and maximum of the data points. P value is determined by Student’s t-test. Significant results are presented as ***P < 0.001.

Fig. 4. The TMZ sensitivity enhanced by BIP-MPC-NP in LN229R cells.

a, b The EdU assay and colony formation assay of LN229R with treatments of TMZ and different nanoinhibitors. The histogram displayed the statistics of EdU assay and colony formation assay, respectively (n = 3). Scale bar = 50 μm. c, d The cell apoptosis assay and the cell-cycle analysis were performed with flow cytometry in LN229R. The histogram displayed the statistics of apoptosis assay and the cell-cycle analysis, respectively (n = 3). The error bars represent the S.D. of three measurements. P value was determined by Student’s t-test. Significant results are presented as *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. BIP-MPC-NP restrains E2F1-mediated DNA damage repair modules via the inhibitory effect of TTP.

a E2F1 binding sites within a region spanning ± 3 kb around TSS in the whole genome. b The signal peaks located in the promoter regions of CHEK1, CHEK2, RAD50, RAD51 and TP53 in E2F1 ChIP-seq data and the binding sites of E2F1 were predicted on JASPAR datasets. The agarose gel electrophoresis displayed the enrichments of E2F1 in the promoter regions of CHEK1, CHEK2, RAD50, RAD51 and TP53 of LN229R. c The luciferase reporter assay displayed the E2F1 transcriptional activity in the promoter regions of CHEK1, CHEK2, RAD50, RAD51 and TP53 in LN229R (n = 3). d The TTP protein expression and E2F1 mRNA expression in LN229R, U87MGR and HG9R treated with MPC-NP or BIP-MPC-NP. e The inhibitory effect of TTP on wild-type or mutant-type ARE motifs within the E2F1 mRNA 3′-UTR in LN229R cells with MPC-NP or BIP-MPC-NP treatments (n = 3). The error bars in c and e represent the S.D. of three measurements. P value is determined by Student’s t-test. Significant results are presented as NS non-significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. The therapeutic response of BIP-MPC-NP combined with TMZ in xenografts tumor derived from TMZ-resistant glioma cells.

a The diagram showed the process of the establishment of models, drug administration and MRI scan. T2-weighted MR images of mouse head at selected times post treatment with TMZ and different nanoinhibitors. The tumor sizes were visualized by H&E staining image. b Tumor volumes were measured on selected days post tumor implantation (n = 10). c Kaplan–Meier survival curves of mouse models after corresponding treatments (n = 10). P = 4.33E−10 for the four groups. Control vs TMZ, P = 9.04E−01; Control vs TMZ + EBP, P = 4.22E−04; Control vs TMZ + MBP, P = 3.40E−04; Control vs TMZ + BIP, P = 4.00E−06; TMZ vs TMZ + EBP, P = 4.13E−02; TMZ vs TMZ + MBP, P = 6.20E−05; TMZ vs TMZ + BIP, P = 4.00E−06; TMZ + EBP vs TMZ + MBP, P = 1.57E−01; TMZ + EBP vs TMZ + BIP, P = 1.32E−02; TMZ + MBP vs TMZ + BIP, P = 4.82E−03. The error bars in b represent the S.D. of ten measurements. P value is determined by Student’s t-test or log-rank test. Significant results are presented as *P < 0.05, **P < 0.01 or ***P < 0.001.

Fig. 7. The mechanistic scheme of a dual functionalized BIP-MPC-NP in temozolomide-resistant glioma.

The mechanistic scheme of BIP-MPC-NP attenuating DNA damage repair to enhance TMZ sensitivity by simultaneously mitigating EGFR and MET activation in TMZ-resistant gliomas.