Human embryonic kidney cells (HEK293T) were obtained from Clontech (Mountain View, CA, United States). The human colon cancer cell lines Caco2 and HCT116 were obtained from the American Type Culture Collection. All cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/mL, Gibco), and streptomycin (0.1 mg/ml, Gibco). The cells were cultured under standard conditions of 5% CO₂ at 37°C. Immortalized cbMSCs (T0016; cbMSC-hTERT) were obtained from Applied Biological Materials Inc. (abm, Richmond, BC, Canada), and were maintained in Prigrow III medium (Cat. No. TM003; abm) containing 20% FBS and 4 ng/ml recombinant basic fibroblast growth factor at 37°C in a 5% CO₂ incubator. The liquid was changed every 2–3 days after the suspension cells were discarded.

MSCsurface antigen markers were detected by flow cytometry. Briefly, 1 × 105 cells were washed and resuspended in 200 μL phosphate buffered saline (PBS). Then, the cell suspension was added with fluorescein isothiocyanate (FITC)-conjugated anti-CD73, allophycocyanin (APC)-conjugated anti-CD105, phycoerythrin-conjugated anti-CD34, and APC-conjugated anti-CD45 and incubated in the dark for 15 min. Subsequently, the cell suspension was washed with 2 ml PBS and centrifuged at 1000rpm for 5 min to remove the supernatant. Lastly, the cell suspension was added with 200 μL buffer solution and detected by a CytoFLEX flow cytometer (Beckman Coulter, United States). The expression of NRP-1 on 293T, Caco2, and HCT116 cells were detected using FITC-conjugated anti-human CD304 (neuropilin-1) antibody (Clone 12C2, Biolegend).

The iRGD-Lamp2b fusion gene encodes (N to C) the signal peptide of Lamp2b (1-28aa), GNSTM glycosylation motif (GNSTM), 3 residue spacer (GSG), iRGD peptide (CRGDKGPDC), 10 residue spacer (GSGSGSGGSS), and Lamp2b (exosomal transmembrane protein, 29-410aa). The iRGD-Lamp2b fusion gene was constructed into lentivirus vector pLVX-IRES-Puro and transfected into 293T cells together with packaging plasmids psPAX2 and pMD2G. The cell supernatant was collected and the lentivirus particles (LV-iRGD-Lamp2b) were purified. cbMSCs were then infected by LV-iRGD-Lamp2b: 1 × 10⁵ cbMSCs cells were spread into 24-well plates 18 h before transfection. The next day, the original culture medium was replaced with fresh medium containing 6 μg/ml polybrene and LV-iRGD-Lamp2b with a multiplicity of infection (MOI) of 5. cbMSCs expressing the iRGD-Lamp2b fusion gene (MSC-iRGD) were obtained after continuous culture with 5 μg/ml puromycin (Sigma-Aldrich, United States).

cbMSCs and cbMSC-iRGD cells at were cultured in DMEM with 10% FBS without exosomes. The supernatant of the cell culture (500 ml) was collected and centrifuged at low speed at 300×g for 10 min, then the supernatant was centrifuged at 2000×g for 10 min to remove the dead cells. The remaining supernatant was obtained and centrifuged at 10,000×g for 30 min to remove the cell debris. After that, the supernatant was centrifuged at high speed, 100,000×g for 70 min, and the crude exosome precipitation (containing a small amount of heteroproteins) was obtained. Lastly, the exosomes were resuspended with PBS and centrifuged at 100,000×g for 70 min to obtain pure exosomes. The exosomes’ morphology was identified by transmission electron microscopy (FEI Tecnai G2 Spirit Bio TWIN).

We employed AMOs with the following sequences: 5′GAA​ACC​CAG​ACA​GAC​AAU​GUA​GCU′3, with 2′ O-methyl modification. AMOs or FAM-labeled-miR-221 inhibitor (FAM-AMO) was loaded onto exosomes using electroporation methods. Briefly, 200 μg exosomes and 10 μL AMO were pre-mixed and added into the perforation dish. After electroporation (100 V, 125 μF, 1 ms), the exosome samples were incubated in a cell incubator for 1 h to promote exosome membrane repair. Subsequently, the mixed exosome samples were centrifuged twice at 100,000×g for 70 min each, and the supernatant was discarded. The sediment contained the compound exosomes loaded with miR-221 inhibitor (AMO-Exos), and stored at −80°C for standby use.

Various concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg) of exosomes containing FAM-labeled-anti-miRNA-221 (iRGD-Exo^FAM−221) were added into 293T, Caco2, and HCT116 cells. After culture for 24 h, FAM fluorescence was detected by flow cytometry after washing with PBS twice to assess the uptake efficiency of exosomes by tumor cells. The green FAM fluorescence was observed and photographed under an inverted fluorescence microscope (MF52-N; MSHOT, Guangzhou, China).

We evaluated cell proliferation with a CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega; G1112). We inoculated 3,000 Caco2 and HCT116 cells into flat 96-well plates. After overnight culture, various concentrations of AMO-Exos and their controls (AMO-NCs) were added. We continued to culture them for 24 h, with a small amount of CellTiter 96® AQueous single solution reagent added to the culture pores. After incubation for 4 h, the absorbance at 490 nm was recorded on a microplate reader. The absorbance value measured at 490 nm is directly proportional to the number of living cells in the culture.

Total RNA was extracted using the RNeasy Plus Universal Mini Kit (73,404, QIAGEN, Duesseldorf, Germany) or the miRNeasy Mini Kit (217,004, QIAGEN). Reverse transcription was performed to obtain complementary DNA with the QuantiTect Reverse Transcription Kit (205,311, QIAGEN). Real-time quantitative polymerase chain reaction (PCR) was performed using ABI7500 (7500, ABI, United States). The mRNA and miRNA expression data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6, respectively. The primers used were as follows: miR-221-forward: 5ʹ-CCT GAA ACC CAG CAG ACA A-3ʹ, backward: 5ʹ-CAG GTC TGG GGC ATG AAC-3ʹ. U6-forward: 5ʹ-CTC GCT TCG GCA GCA CA-3ʹ, backward: 5ʹ-AAC GCT TCA CGA ATT TGC GT-3ʹ. NRP-1-forward: 5ʹ-GGC GCT TTT CGC AAC GAT AAA-3ʹ, backward: 5ʹ-TCG CAT TTT TCA CTT GGG TGA T-3ʹ. GAPDH-forward: 5ʹ-GGA GCG AGA TCC CTC CAA AAT-3ʹ, backward: 5ʹ-GGC TGT TGT CAT ACT TCT CAT GG-3ʹ.

A total of 700 cells were pre-inoculated in a 6-well plate. After overnight culture, 20 μg exosomes containing AMO-Exos or AMO-NCs were added to Caco2 and HCT116 cells for another 14 days. When obvious cell cloning was observed in the culture dish, the culture medium was abandoned, and the clones were fixed with formaldehyde for 15 min, stained with gentian violet for 30 min, and the number of clones was determined under the microscope. Each experiment was repeated 3 times.

We purchased 4-week-old B-NDG (NOD-Prkdc^scid Il2rg^tm1/Bcgen) female mice from Biocytogen (Biocytogen Co., Ltd., Beijing, China) and raised them in an SPF grade environment. After 7 days of adaptive feeding, the mice were subcutaneously inoculated with HCT116-luc cells (stably expressing firefly luciferase). Ten days after inoculation, obvious xenograft formation could be seen. Ten minutes after intraperitoneal injection of D-Luciferin (3 mg/mouse), the mice were imaged, and the fluorescence values were analyzed. The mice were randomly divided into 3 groups (n = 3) according to the fluorescence values, and received intratumoral injection with AMO-Exos (200 μg, 50 μL), NC-Exos (200 μg, 50 μL), or PBS (50 μL) on day 0, day 3, and day 7, respectively. In vivo imaging was performed on the mice every 7 days. The size of the transplanted tumor was measured with vernier caliper twice a week, and the mental state and diet of the mice were observed daily.

Data drawing and analysis were performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, United States). The data were described as mean ± standard error of the mean. The analysis of data between groups was performed using an analysis of variance or a t-test. Tumor growth in the mice was compared using Mauchly’s test of sphericity.

We first identified the morphology and molecular markers of the cbMSCs. As shown in Figure 1A, under the light microscope, the cbMSCs were spindle-shaped and wall-attached. The flow cytometry results showed that the specific cbMSC surface markers, CD73 and CD105, were highly expressed in the cbMSCs, whereas the levels of CD34 and CD45, the specific surface markers of hematopoietic stem cells, were low (Figure 1B). In order to obtain iRGD peptide modified exosomes (iRGD-Exos), plasmids containing the iRGD-Lamp2b fusion gene were further transferred into cbMSCs using lentivirus (Figure 1C). Exosomes from iRGD-modified cbMSCs were collected and purified, and the AMOs or corresponding NCs were loaded into the exosomes by electric transduction. Subsequently, the iRGD peptide was bound to NRP-1, a receptor protein of tumor cells, to mediate the anti-tumor effect.

AMO-loaded exosomes were taken up by colon cancer cells through NRP-1.

The morphological identification of AMO-Exos was performed by transmission electron microscopy (Figure 2A). Considerable research has shown that the residual peptide, CRGDK/R, the product of iRGD hydrolyzation, is the specific ligand of NRP-1, which is highly expressed in tumor tissues. As shown in Figure 2B, the expression levels of NRP-1 in various tumors were analyzed. The results suggested that NRP-1 was highly expressed in malignant melanoma, breast cancer, liver cancer, and colon cancer. Quantitative PCR and flow analysis were performed, and the results showed that NRP-1 was highly expressed in the colon cancer cells Caco2 and HCT116, rather than the 293T cells (Figures 2C,D). To investigate whether iRGD-Exos could combine with NRP-1-positive colon cancer cells and deliver AMO into tumor cells, AMO was pre-labeled with fluorescein amidite (FAM) before being loaded into exosomes, and was co-cultured with human colon cancer cell lines and 293T cells. The results showed that FAM green fluorescence was increased in Caco2 and HCT116 cells in a dose-dependent manner (Figure 2E), indicating that exosomes containing FAM-AMO were taken up by colon cancer cells. Furthermore, 4 h after incubation, we observed strong green fluorescence in the HCT116 cells, but almost no fluorescence in the 293T cells (Figure 2F).

We further investigated the anti-tumor function of exosomes loaded with AMO-Exos. Compared with the exosomes loaded with the NC-Exos, AMO-Exos could significantly downregulate the level of miRNA-221 in colon cancer cells Caco2 and HCT116 (Figure 3A). The results showed that AMO-Exos significantly inhibited cell proliferation, whereas no significant inhibition was observed under NC-Exo treatment (Figure 3B). Furthermore, AMO-Exos markedly suppressed the clonogenic abilities of Caco2 and HCT116 (Figures 3C,D).

Subcutaneous HCT116 tumor-bearing mice were established to investigate the potential anti-tumor effects of AMO-Exos in vivo. When the tumors formed, the mice were divided into 3 groups and received intratumoral treatment with NC-Exos or AMO-Exos 3 times (at days 0, 3, and 7). The mice were imaged in vivo using bioluminescent imaging every 7 days and tumor volume was measured. The results indicated that the fluorescence intensity of the mice was significantly suppressed after AMO-Exo treatment (Figure 4A), and the tumor volume also grew significantly more slowly than the control group (Figure 4B). Quantitative PCR detection of the xenograft revealed that AMO-Exo treatment significantly reduced the expression of miR-221 (Figure 4C), suggesting that anti-miR-221 transferred by exosomes acted as a tumor suppressor in vivo. Further analysis of the expression of downstream genes of miR-221 showed that the expression of several genes (BMF, CDKN1B, CDKN1C, PTEN, TIMP3, and MET) was significantly upregulated after AMO-Exo treatment (Figure 4D).