Engineering a HEK-293T exosome-based delivery platform for efficient tumor-targeting chemotherapy/internal irradiation combination therapy

Abstract

Exosomes are nanoscale monolayer membrane vesicles that are actively endogenously secreted by mammalian cells. Currently, multifunctional exosomes with tumor-targeted imaging and therapeutic potential have aroused widespread interest in cancer research. Herein, we developed a multifunctional HEK-293T exosome-based targeted delivery platform by engineering HEK-293T cells to express a well-characterized exosomal membrane protein (Lamp2b) fused to the αv integrin-specific iRGD peptide and tyrosine fragments. This platform was loaded with doxorubicin (Dox) and labeled with radioiodine-131 (¹³¹I) using the chloramine-T method. iRGD exosomes showed highly efficient targeting and Dox delivery to integrin αvβ3-positive anaplastic thyroid carcinoma (ATC) cells as demonstrated by confocal imaging and flow cytometry in vitro and an excellent tumor-targeting capacity confirmed by single-photon emission computed tomography-computed tomography after labeling with ¹³¹I in vivo. In addition, intravenous injection of this vehicle delivered Dox and ¹³¹I specifically to tumor tissues, leading to significant tumor growth inhibition in an 8505C xenograft mouse model, while showing biosafety and no side effects. These as-developed multifunctional exosomes (denoted as Dox@iRGD-Exos-¹³¹I) provide novel insight into the current treatment of ATC and hold great potential for improving therapeutic efficacy against a wide range of integrin αvβ3-overexpressing tumors.

Keywords: Anaplastic thyroid carcinoma; Combination therapy; Exosome; Radioiodine-131; Tumor targeting; iRGD peptide.

Scheme 1

A Chemical structure of the iRGD peptide. B Chemical structure of Dox. C Schematic illustration of the procedure to produce engineered HEK-293T exosomes (donated as Dox@iRGD-Exos-¹³¹I). D Schematic representation of Dox@iRGD-Exos-¹³¹I for efficient chemotherapy combined with ¹³¹I labeling. Making full use of lentiviral vector technology and the structure of the exosomal membrane, we developed an engineered HEK-293T exosome-based delivery system that intelligently integrates three functions: tumor targeting, ¹³¹I and Dox coloading with high payloads, and tumor-targeted therapy. After intravenous injection, Dox@iRGD-Exos-¹³¹I efficiently accumulated at the tumor site, resulting in a significantly enhanced antitumor chemo/internal irradiation combination therapy effect

Fig. 1

Expression level of integrin αvβ3 in ATC. A Immunohistochemistry of the paraffin-embedded human ATC cancerous tissues and paracancerous normal tissues to determine the expression of integrin αvβ3 (n = 8, 16 samples, scale bar = 10 μm). B Western blotting analysis of integrin αvβ3 expression in ATC cell lines (Hth7, Cal-62, THJ16T, and 8505C) and a normal thyroid cell line. Gray analysis was performed by ImageJ, *P < 0.05

Fig. 2

Characterization of Dox@iRGD-Exos-¹³¹I. A The main composition of the EGFP-C1-iRGD-Tyr7-Lamp2b plasmid and an image of iRGD/blank-Tyr7-EGFP-293T cells using fluorescence microscopy (scale bar = 100 μm). Representative TEM images and particle size distribution of B blank-Exos, C iRGD-Exos, E blank-Exos-¹³¹I, F iRGD-Exos-¹³¹I and G Dox@iRGD-Exos-¹³¹I (scale bar = 200 nm). D Western blotting analysis of exosome marker proteins (TSG101, CD9 and Alix) of blank-Exos and iRGD-Exos

Fig. 3

In vitro targeting of iRGD-Exos. A Confocal microscopy images of 8505C cells incubated with PKH26-blank-Exos and PKH26-iRGD-Exos at 4 h. Nuclei were stained with DAPI (blue). Fluorescence from PKH26 (red) and DAPI (blue) was observed. The scale bar is 10 μm. B Flow cytometric analysis of PKH26-iRGD-Exos binding to 8505C cells. Exosomes were labeled with PKH26 and incubated with 8505C for different lengths of time (3 h, or 6 h). ***P < 0.001

Fig. 4

Cell viability assay. Viability of A Nthy-ori 3-1 and B 8505C cells treated with different concentrations of iRGD-Exos (0–1000 μg/mL) for 24 h. C 8505C and D Hth7 cells were incubated with control medium, iRGD-Exos, Na¹³¹I (3.7 MBq/well), blank-Exos-¹³¹I, iRGD-Exos-¹³¹I, Dox, Dox@iRGD-Exos, or Dox@iRGD-Exos-¹³¹I (15 μg/mL Dox) at 24 h at the same dose of radioactivity (3.7 MBq/well). A CCK-8 assay was used to assess cell viability in each group. NS (not significant) indicates P > 0.05 compared to the control group; *P < 0.05; **P < 0.01; $ indicates P < 0.01 compared to the other groups; & indicates P < 0.001 compared to the other groups

Fig. 5

A In vivo fluorescence imaging of 8505C tumor-bearing nude mice at 0 h, 1 h, 8 h and 24 h after tail vein administration of DiR-labeled blank-Exos/iRGD-Exos. Black circle indicates the tumor site. B Quantitative analysis of the tumor fluorescence intensity of the in vivo images. C In vivo SPECT/CT imaging of 8505C tumor-bearing nude mice after intravenous injection of different drug combinations (Na¹³¹I, blank-Exos-¹³¹I, and iRGD-Exos-¹³¹I) at 0.5 h, 24 h, 72 h and 96 h postinjection. White circle indicates the tumor site. D Quantitative analysis of the radiation counts at the tumor site. *P < 0.05, **P < 0.01, ***P < 0.001. # indicates P < 0.01 compared to the Na¹³¹I group. & indicates P < 0.001 compared to the Na¹³¹I group

Fig. 6

Antitumor efficacy in vivo. A Growth curves of the 8505C tumors after the mice were injected with PBS, iRGD-Exos, Na¹³¹I, blank-Exos-¹³¹I, iRGD-Exos-¹³¹I or Dox@iRGD-Exos-¹³¹I. Tumor volume was measured every 3 days until the end of the observation period. B Ex vivo image of the tumors from the sacrificed mice at 18 days postinjection. C Quantitative analysis of the tumor weights from the mice in the different treatment groups. D Changes in body weights of the different drug-treated mice during the observation period. Data are presented as the mean ± SD. *P < 0.05, ***P < 0.001

Fig. 7

Representative H&E-stained images of the tumors and major organs (heart, liver, spleen, lung, and kidney) collected from 8505C tumor-bearing mice in different treatment groups after sacrifice. The scale bar represents 10 μm