Engineered exosomes for targeted co-delivery of miR-21 inhibitor and chemotherapeutics to reverse drug resistance in colon cancer

Abstract

Background: 5-Fluorouracil (5-FU) has been commonly prescribed for patients with colorectal cancer (CRC), but resistance to 5-FU is one of the main reasons for failure in CRC. Recently, microRNAs (miRNAs) have been established as a means of reversing the dilemma by regulating signaling pathways involved in initiation and progression of CRC. However, how to safely and effectively deliver miRNA to target cells becomes a main challenge.

Results: In this study, Engineered exosomes were exploited to simultaneously deliver an anticancer drug 5-FU and miR-21 inhibitor oligonucleotide (miR-21i) to Her2 expressing cancer cells. Purified engineered exosomes from the donor cells loaded with 5-FU and miR-21i via electroporation to introduce into 5-FU-resistant colorectal cancer cell line HCT-116^5FR. Furthermore, systematic administration of 5-FU and miR-21i loaded exosomes in tumor bearing mice indicated a significantly anti-tumor effect. The results showed that the engineered exosome-based 5-FU and miR-21i co-delivery system could efficiently facilitate cellular uptake and significantly down-regulate miR-21 expression in 5-FU resistant HCT-116^5FR cell lines. Consequently, the down-regulation of miR-21 induced cell cycle arrest, reduced tumor proliferation, increased apoptosis and rescued PTEN and hMSH2 expressions, regulatory targets of miR-21. Of particular importance was the significant reduction in tumor growth in a mouse model of colon cancer with systematic administration of the targeting miR-21i. More excitedly, the combinational delivery of miR-21i and 5-FU with the engineered exosomes effectively reverse drug resistance and significantly enhanced the cytotoxicity in 5-FU-resistant colon cancer cells, compared with the single treatment with either miR-21i or 5-FU.

Conclusion: The strategy for co-delivering the functional small RNA and anticancer drug by exosomes foreshadows a potential approach to reverse the drug resistance in CRC and thus to enhance the efficacy of the cancer treatment.

Keywords: 5-FU; Delivery system; Drug resistance; Exosomes; miR-21 inhibitor.

Scheme 1

Engineered exosomes based nanocarrier for 5-FU and miR-21i simultaneously deliver to HCT-116^5FR human colon cancer cells for enhancing chemotherapy efficacy

Fig. 1

Isolation and molecular characterization of engineered exosomes. a Schematics of DNA constructs used for the production of fusion proteins LG (upper) and THLG (lower). b Fluorescence images of the GFP, LG and THLG in 293T cells ( ×400). c Biomarkers of exosomes were determined by western blot analysis of exosomes. d Western blot analysis of GFP in exosomes obtained from supernatants of GFP-293T cells, LG-293T cells and THLG-293T cells. e Confocal microscopy of exosomes isolated from 0.22 filtered conditioned medium of GFP-293T cells, LG-293T cells and THLG-293T cells ( ×1000). f Transmission electron micrograph of THLG-EXO and THLG-EXO/5-FU/miR-21i. g Size distribution of THLG-EXO and THLG-EXO/5-FU/miR-21i measured by DLS

Fig. 2

The cellular tropism of THLG-EXO in vitro. a Schematics of DNA constructs used for the production of fusion proteins Her2-mCherry (upper panel); flow cytometric analyses of Her2 from SGC-7901 WT cells and Her2-mcherry-SGC-7901 cells with a FITC labeled anti-Her2 monoclonal antibody (lower panel). b Confocal microscopy images of cellular uptake of THLG-EXO or LG-EXO after 3 h incubation with SGC-7901 WT cells and Her2-mcherry-SGC-7901 cells co-culture model. red shows Her2-mcherry-SGC-7901 cells. Green represents THLG-EXO or LG-EXO. Right panel shows enlarged graphs with frame indicating internalized exosomes in cells (×400). c Flow cytometry analysis of co-culture cells after incubation with THLG-EXO or LG-EXO (upper panel); d quantification of exosomes internalization based on flow cytometry analysis (lower panel). Data are expressed as mean ± SD. n = 5; **p < 0.01

Fig. 3

The results of HCT-116^5FR cells treated with THLG-EXO/5-FU/miR-21i in vitro. a Real-time fluorescent quantitative PCR analysis of relative expression change of miR-21 level in HCT-116^5FR cells treated with THLG-EXO/5-FU/miR-21i, THLG-EXO or miR-21i. Data were expressed as mean ± SD. n = 3. b Western blot analysis of hMSH2 and PTEN protein levels of HCT-116^5FR cells treated with THLG-EXO/5-FU/miR-21i. c Flow cytometric analysis of AnnexinV-FITC/PI stained HCT-116^5FR cells treated with THLG-EXO, THLG-EXO/miR-21i, THLG-EXO/5-FU or THLG/5-FU/miR-21i. d Cell cycle profiles of HCT-116^5FR cells after treatment as described in c; e the proliferation of HCT-116^5FR cells after treatment as described in c. Data were expressed as means ± SD. n = 5;*: significantly different from other groups (p < 0.05), **: extremely significantly different from other groups (p < 0.01)

Fig. 4

Tumor targeting ability of THLG-EXO in a nude mouse xenograft model using HCT-116^5FR cells. a In vivo fluorescent images of HCT-116^5FR-bearing nude mice after intravenous injection of DiR-labeled THLG-EXO (left panel) or LG-EXO (right panel). b Quantitative analysis showed the relative fluorescence intensities in the tumor region (upper panel) and the liver (lower panel) following administration of THLG-EXO or LG-EXO. Data were expressed as means ± SD. n = 5; *P < 0.05, **P < 0.01 versus the LG-EXO group at the same time point

Fig. 5

Antitumor activity of THLG-EXO/5-FU/miR-21i in nude mice xenograft using HCT-116^5FR cells. a Representative bioluminescent images of tumor growth in nude mice treated with THLG-EXO, THLG-EXO/miR-21i, THLG-EXO/5-FU or THLG-EXO/5-FU/miR-21i at different time point. Signals were adjusted to the same color scale for the entire time course. b The mean bioluminescence intensity (BLI) for each mouse/group, line chart represented quantification of the relative bioluminescent intensity in tumor site. c Tumor weights of different treated groups. T1, T2, T3 and T4 represented the treatment of THLG-EXO, THLG-EXO/miR-21i, THLG-EXO/5-FU, THLG-EXO/5-FU/miR-21i, respectively. d TUNEL assessments of tumor tissues treated with THLG-EXO, THLG-EXO/miR-21i, THLG-EXO/5-FU, THLG-EXO/5-FU/miR-21i, respectively. e Western blot of hMSH2 and PTEN in the tissue of tumor region were performed for 24 h after administration. Data were expressed as means ± SD. n = 6; *P < 0.05, **P < 0.01 versus the THLG-EXO groups or THLG-EXO/miR-21i group, and ^#P < 0.05 versus the THLG-EXO/5-FU groups

Fig. 6

The systematic toxicity assessment of THLG-EXO. Histopathological analysis of heart, lung, liver, kidney and spleen sections stained with hematoxylin and eosin of BALB/c mice post-intravenous injection of 20 mg/kg THLG-EXO or PBS for 7 days (one dose every other day). Images were obtained under Nikon Ti microscope using a  × 40 objective