Engineered extracellular vesicles with polypeptide for targeted delivery of doxorubicin against EGFR‑positive tumors

Abstract

Lack of effective tumor‑specific delivery systems remains an unmet clinical challenge for the employment of chemotherapy using cytotoxic drugs. Extracellular vesicles (EVs) have recently been investigated for their potential as an efficient drug‑delivery platform, due to their good biodistribution, biocompatibility and low immunogenicity. In the present study, the formulation of GE11 peptide‑modified EVs (GE11‑EVs) loaded with doxorubicin (Dox‑GE11‑EVs), was developed to target epidermal growth factor receptor (EGFR)‑positive tumor cells. The results obtained demonstrated that GE11‑EVs exhibited highly efficient targeting and drug delivery to EGFR‑positive tumor cells compared with non‑modified EVs. Furthermore, treatment with Dox‑GE11‑EVs led to a significantly inhibition of cell proliferation and increased apoptosis of EGFR‑positive tumor cells compared with Dox‑EVs and free Dox treatments. In addition, it was observed that treatment with either free Dox or Dox‑EVs exhibited a high level of cytotoxicity to normal cells, whereas treatment with Dox‑GE11‑EVs had only a limited effect on cell viability of normal cells. Taken together, the findings of the present study demonstrated that the engineered Dox‑GE11‑EVs can treat EGFR‑positive tumors more accurately and have higher safety than traditional tumor therapies.

Keywords: EGFR‑positive cancer; chemotherapy; colorectal cancer; engineered extracellular vesicles; glioma; targeted drug delivery.

Figure 1.

Extraction diagram of Dox-GE11-EVs and plasmid profile of CD63-GE11. (A) Diagram of preparation of Dox-GE11-EVs and the target model. (B) Construction of recombinant plasmid carrying CD63-GE11. Dox, doxorubicin; EVs, extracellular vesicles.

Figure 2.

Establishment and characterization of HEK-293T-GE11 cells. (A) Nucleic Acid Gel Electrophoresis analysis of CD63-GFP fragment and CD63-GE11 fragment in 293T cells, and 293T-transfected cells. (B) Representative fluorescence images of GFP in 293T-GE11 cells. Scale bar, 100 µm. (C) Western blot analysis of CD63, GFP, GM130 and β-actin expression in 293T cells, blank EVs, EVs and GE11-EVs. EVs, extracellular vesicles.

Figure 3.

Preparation and characterization of Dox-GE11-EVs. (A) Western blot analysis of CD63, Alix, GM130 and β-actin expression in 293T cell and EVs. (B) TEM images of EVs. Scale bar, 100 nm. (C) Size distribution analysis of EVs using NTA. (D) Standard curve of Dox using high-performance liquid chromatography. (E) TEM images of Dox-GE11-EVs. Scale bar, 100 nm. (F) Size distribution analysis of Dox-GE11-EVs using NTA. (G) Confocal images and co-localization analysis between Dox (red) and PKH67-labeled GE11-EVs (green). Scale bar,10 µm. Dox, doxorubicin; EVs, extracellular vesicles; TEM, transmission electron microscopy; LE, drug loading efficiency; EE, encapsulation efficiency.

Figure 4.

Cellular uptake of GE11-EVs in CRC cells in vitro. (A) Western blot analysis of EGFR expression in eight CRC cell lines (HCT116, DLD1, Lovo, RKO, HCT15, SW480, SW620 and Caco2). (B) Representative confocal images of the cellular uptake of GE11-EVs in CRC cells. Scale bar, 50 µm. (C) Flow cytometric analysis of the cellular uptake of GE11-EVs in CRC cells. The control group was treated with PBS. (D) Quantitative flow cytometric analysis of the cellular uptake of GE11-EVs in CRC cells. **P<0.01 (unpaired Student's t-test). EVs, extracellular vesicles; CRC, colorectal cancer; ns, not significant.

Figure 5.

Cellular uptake of GE11-EVs in GBM cells in vitro. (A) Western blot analysis of EGFR expression in eight GBM cell lines (U251, SF763, LN229, A172, LN18, U118, U-87MG and LNZ308). (B) Representative confocal images of the cellular uptake of GE11-EVs in GBM cells. Scale bar, 50 µm. (C) Flow cytometric analysis of the cellular uptake of GE11-EVs in GBM cells. The control group was treated with PBS. (D) Quantitative flow cytometric analysis of the cellular uptake of GE11-EVs in GBM cells. **P<0.01 (unpaired Student's t-test). EVs, extracellular vesicles; GBM, glioblastoma; ns, not significant.

Figure 6.

Dox-GE11-EVs promote anticancer efficiency to EGFR-positive cells and limit cytotoxicity to normal cells. (A) Viability was examined by Cell Counting Kit-8 assays in EGFR-positive and normal cells with DOX from different sources for 24 (up) or 48 h (down). The Dox concentration used by HCT116 and NCM460 was 2.0 µg/ml, Dox concentration used by SF763 and HA1800 was 0.25 µg/ml. *P<0.05 and **P<0.01 (One-way ANOVA). (B) Western blot analysis of p-H2AX and Cleaved Caspase-3 expression in EGFR-positive and normal cells with different treatments for 24 h. Control group was not treated. (C) TUNEL staining of apoptotic cells in EGFR-positive and normal cells with different treatments for 24 h. Scale bar, 100 µm. Control group was not treated. Dox, doxorubicin; EVs, extracellular vesicles; ns, not significant.