Targeted delivery of Dbait by an artificial extracellular vesicle for improved radiotherapy sensitivity of esophageal cancer

Abstract

Intensification of radiotherapy has been shown to be an effective way for improving the therapeutic efficacy of radiation sensitive malignancies such as esophageal cancer (EC). The application of DNA Bait (Dbait), a type of DNA repair inhibitor, is an emerging strategy for radiosensitization. In this study, a Eca-109 cancerous cytomembrane-cloaked biomimetic drug delivery system (DDS), CMEC-Dbait, was designed and successfully fabricated, for targeted delivery of Dbait. Our systematic evaluation demonstrated that the ingenious artificial gastrointestinal extracellular vesicle owns neat spherical structure, proper particle size (154.6±5.5 nm) and surface charge (2.6±0.3 mV), favourable biocompatibility and immunocompatibility, being conducive to in vivo drug delivery. Besides, Eca-109 cytomembrane coating endowed CMEC-Dbait with effective targeting ability to homologous EC cells. Owing to these advantages, the biomimetic DDS was proved to be a potent radiosensitizer in vitro, indicated by remarkably reduced cell viability and enhanced cellular apoptosis by the combination therapy of radiation and CMEC-Dbait. The result was validated in vivo using mouse xenograft models of EC, the results illustrated that radiotherapy plus CMEC-Dbait significantly suppressed tumor growth and prolonged survival of tumor bearing mice. Western blotting results showed that CMEC-Dbait can significantly inhibit DNA damage repair signaling pathways by simulating DNA double-strand breaks both in and ex vivo. In conclusion, the versatile biomimetic CMEC-Dbait was characterized of low toxicity, excellent biocompatibility and satisfactory drug delivery efficiency, which is confirmed to be an ideal radiosensitizer for homologous cancer and merits further investigation in both pre-clinical and clinical studies.

Keywords: Dbait; Esophageal cancer; artificial gastrointestinal extracellular vesicle; biomimetic drug delivery system; radiosensitivity.

Figure 1

Schematic illustration of CCM-cloaked biomimetic vesicles for enhanced radiosensitivity of homotypic EC cells.

Figure 2

Characterization of the biomimetic vesicles (CMEC-Dbait). (A) TEM morphology of CMEC-Dbait NPs. Scale bar: 500 nm. (B-D) Size distribution (B), Hydrodynamic diameters (C), and Zeta potential (D) of the artificial extracellular vesicles (CMEC-Dbait) and counterparts. (E) UV-vis absorption of CMEC-Dbait and counterparts. (F) SDS-PAGE for different samples (I: Eca-109 cell lysate, II: Eca-109 cell membrane, III: empty CMEC vehicles IV: CMEC-Dbait).

Figure 3

Serum stability and controlled drug release profiles of CMEC-Dbait. A. Excellent in vitro serum stability of CMEC-Dbait. B. Drug release profile of Dbait from biomimetic DDS at different conditions.

Figure 4

In vitro biocompatibility of the biomimetic DDS. A. Hemolysis quantification of RBC incubated with different formulations of Dbait. B. Cytotoxicity of free and CMEC encapsulated Dbaits. C. CLSM images of J774A.1 macrophages incubated with different formulations of QDs. Green and blue fluorescence respectively indicates QDs and nucleus. Scale bar: 50 µm.

Figure 5

Cellular uptake of the biomimetic DDS. A. Effects of CCM camouflaging on intracellular uptake of QDs in homologous cells (Eca-109 and KYSE-150) by FCM. B. Quantitative analysis of FCM results. MFI was counted using FlowJo software. Data are mean ± SD (n=3), **P < 0.01. C. Effects of CCM camouflaging on the intracellular uptake of QDs in homologous cells by CLSM. Green: QD; blue: nucleus. Scale bar: 20 µm.

Figure 6

CMEC-Dbait improved in vitro radiosensitization of EC cells. (A, B) Cell viability of Eca-109 (A) and KYSE-150 (B) cells after treatment with different formulations of Dbait and radiation (0-8Gy) by CCK-8 analysis. (C, D) Apoptosis of Eca-109 (A) and KYSE-150 (B) cells after different treatment analyzed by Annexin V/PI staining. (E) Live/dead staining of Eca-109 and KYSE-150 cells with different treatments. For necrotic cells, the reactive dye can permeate the compromised membranes and react with free amines both in the interior and on the cell surface. In contrast, only the cell-surface amines of viable cells are available to react with the dye, resulting in relatively dim staining. Scale bar: 50 µm. Data expressed as means ± SD. *P < 0.05; **P < 0.01. Scale bar: 50 µm.

Figure 7

A. Western Blotting analysis of γ-H2AX, DNA-PKcs and PARP in Eca-109 and KYSE-150 cells being treated with radiation (4 Gy) plus different formulations of Dbait. B. Gray value of WB protein bands analyzed by Quality One software (Version 4.62) displayed in heat map.

Figure 8

CMEC-Dbait is an effective radiosensitizer in vivo. (A, B) Mice bearing Eca-109 xenograft tumors were injected with different formulations of Dbait or PBS, followed by radiotherapy or not. Tumor growth curves (n=3) (A) and survival curves (n=5) (B) were generated. Data expressed as means ± SD. (C) WB analysis of γ-H2AX, DNA-PKcs and PARP in xenograft tumors being treated with radiation plus different formulations of Dbait. (D) Gray value of WB protein bands analyzed by Quality One software (Version 4.62) displayed in heat map. (E-G) Blood routine analysis of tumor-bearing mice after different treatment. (H-K) Biochemical indexes in plasma taken from tumor bearing mice after treatment. ALT: Alanine Aminotransferase; ALP: Alkaline Phosphatase; BUN: Blood Urea Nitrogen; CREA: serum Creatinine. Data expressed as violin plot.