2023 Volume 71 Issue 11 Pages 819-823
Exosomes are a type of extracellular vesicles that contain diverse molecules and are present in our body fluids. They play a crucial role in transporting materials and transmitting signals between cells. Currently, there have been numerous reports on the use of exosomes in drug delivery systems (DDS). However, most existing methods for utilizing exosomes in DDS require the isolation and purification of exosomes, which raises concerns about yield and potential damage to the exosomes. Recently, we have developed a novel DDS called “ExomiR-Tracker” that harnesses exosomes without the need for isolation and purification. This system aims to deliver nucleic acid drugs effectively. ExomiR-Tracker consists of an anti-exosome antibody equipped with nona-D-arginines (9 mer) and nucleic acid drugs which have complementary sequence of target microRNA (anti-miR). In this study, we modified ExomiR-Tracker by incorporating branched nona-D-arginines (9 + 9 mer) molecules (referred to as Branch ExomiR-Tracker) and evaluated its efficacy in lung adenocarcinoma cells (A549 cells). The improved complex formation ability and enhanced cellular uptake of anti-miR, demonstrated by our findings, highlight the advantages of incorporating branched oligoarginine peptides into the ExomiR-Tracker platform. These results represent significant progress in revealing the effectiveness of Branch ExomiR-Tracker against adhesive cancer cells, which has not been shown to be effective with the conventional Linear ExomiR-Tracker.
Exosomes are small lipid bilayer extracellular vesicles with a size range of 30–150 nm that circulate within our body fluids. These vesicles contain a variety of molecules, including microRNAs (miRNAs), DNAs, and proteins.1) Initially, exosomes were believed to be vesicles involved in the extracellular secretion of waste products. However, as more molecules were discovered within exosomes and their roles in substance transportation and intercellular signal transmission were elucidated, they garnered attention as a natural drug delivery system (DDS).2–5) In recent years, numerous studies have explored the use of exosomes as carriers for nucleic acid drugs and small molecules, capitalizing on their unique properties.6–9) Nonetheless, employing exosomes for DDS necessitates the isolation and purification of exosomes from cell culture supernatants or blood samples, a process that poses challenges such as low yield and the risk of exosome denaturation.10–12)
To address these difficulties, we have recently developed a novel DDS called “ExomiR-Tracker” that bypasses the need for isolating and purifying exosomes.13) This system utilizes an anti-exosome antibody (anti-Exo) combined with cationic nona-D-arginines (9 mer) and antisense oligonucleotides that target microRNAs (anti-miR). Our study demonstrated that ExomiR-Tracker attaches to the surface of exosomes and is simultaneously internalized into recipient cells along with the exosomes. We also verified that the intracellular uptake and gene regulatory activity of ExomiR-Trackers in oral squamous carcinoma cells (Cal27 cells) are dependent on exosomes. Furthermore, we achieved successful delivery of small interfering RNAs (siRNAs) using this exosome-hijacking system into multiple myeloma cells, which are a type of hematologic tumor.14) To deliver siRNAs, we employed anti-Exo combined with branched nona-D-arginines (9 + 9 mer), and this complex with branched nona-D-arginines exhibited higher binding affinity to siRNA compared to the conventional linear form.14)
Based on our previous findings, we conducted a study to develop and evaluate the efficacy of ExomiR-Tracker, which consists branched-oligoarginines-introduced anti-Exo and anti-miR (Branch ExomiR-Tracker), in targeting solid tumor cells. As a representative model cell for solid tumors, we selected human lung adenocarcinoma A549 cells, as lung cancer is a challenging solid cancer that requires novel therapeutic approaches. MicroRNA-21 (miR-21), which is highly expressed in A549 cells and linked to oncogenesis, was selected as the target microRNA.15–19) To specifically target miR-21, we prepared Branch ExomiR-Tracker by equipping it with anti-miR-21, which has a sequence that complements miR-21. We then assessed the intracellular uptake of Branch ExomiR-Tracker and its ability to functionally inhibit miR-21 in comparison to the conventional ExomiR-Tracker (Linear ExomiR-Tracker) in A549 cells. The results of our study will provide fundamental information for the design of ExomiR-Tracker for therapeutic applications in lung cancer cells.
Initially, we examined the intracellular uptake of anti-Exo in A549 lung adenocarcinoma cells. Based on previous research,13) to target the antigens on the exosome membrane, CD63, CD9, and CD81, which are tetraspanin transmembrane proteins,20,21) were selected. Additionally, TSG101, a well-known exosomal protein,22) was selected as a control. These antibodies were labeled with Alexa Fluor 647 (AF647) and were introduced into the culture medium of A549 cells. After 24 h of incubation, the cells were fixed, stained, and examined using confocal microscopy to analyze the uptake of the antibodies (Fig. 1-A). Our observations revealed that the anti-CD63 antibody demonstrated effective uptake into A549 cells. Conversely, the cellular uptake of anti-CD9 and anti-CD81 antibodies was significantly lower compared to that of the anti-CD63 antibody. Similar results were also observed in Cal27 cells (human oral squamous carcinoma) and HeLa cells (human cervical carcinoma).13)
(A) Confocal imaging of fluorescent-labeled anti-exosome antibodies in A549 cells after 24 h of incubation. [anti-exosome antibody] = 300 nM. (B) Quantification of fluorescence intensity at 633 nm. Obtained images (n = 3) were analyzed by ImageJ and the fluorescence intensity was corrected by the number of cells. Data are expressed as the mean ± standard deviation (S.D.). The experiments were performed in duplicate.
Furthermore, we investigated whether internalization of the anti-CD63 antibody occurred in an exosome-dependent manner. To examine this, A549 cells were exposed to the anti-CD63 antibody and incubated with or without additional exosomes in a serum-free medium. After 12 h of incubation, the cellular uptake of the antibody was analyzed using confocal microscopy (Fig. 2-A). The results revealed that the fluorescence intensity of A549 cells co-incubated with both the anti-CD63 antibody and exosomes was approximately 1.7 times higher than that observed in cells without exosomes (Fig. 2-B). These findings indicate that the anti-CD63 antibody specifically binds to CD63 on the exosome surface and is intracellularly delivered with exosomes. Consequently, based on these results, we selected the anti-CD63 antibody as the anti-Exo of Branch ExomiR-Tracker for targeting lung adenocarcinoma cells.
(A) Confocal imaging of fluorescent labeled anti-CD63 antibodies in A549 cells after 12 h of incubation with or without A549 exosomes. The final concentration of antibody and exosome were 300 nM and 15 µg/well, respectively. (B) Quantification of fluorescence intensity at 633 nm. Obtained images (n = 3) were analyzed by ImageJ and the fluorescence intensity was corrected by the number of cells. Data are expressed as the mean ± S.D. The experiments were performed in duplicate.
According to a previous study,13) we performed a modification of the anti-CD63 antibody by introducing nona-D-arginines (Fig. 3). Initially, the antibody was treated with Traut’s Reagent (2-iminothiolane HCl) for 1 h at 20 °C, resulting in the production of a thiolated antibody (immunoglobulin G (IgG)-SH). Subsequently, IgG-SH was combined with either a linear oligoarginine peptide (9 mer) or branched oligoarginine peptide (9 + 9 mer) and incubated at 4 °C. This process generated a linear arginylated antibody (IgG-9r) and branched arginylated antibody (IgG-9r(9r)), respectively. The stoichiometry of thiol modification on the anti-Exo was calculated based on the signals resulting from reaction with Ellman’s reagent (Supplementary Fig. S1). The introduction number of linear oligoarginine peptide and branched oligoarginine peptide to IgG was estimated to be 3.59 and 3.57, respectively.
Plain: 2′-O-methyl RNA, Underlined: locked-nucleic acid (LNA), R: TAMRA.
Finally, the arginylated antibodies and anti-miRs were mixed in phosphate buffered saline (PBS) and incubated for 20 min at 20 °C, resulting in the formation of ExomiR-Trackers. The functional assessments of ExomiR-Trackers were conducted using an electrophoretic mobility shift assay (EMSA) (Supplementary Fig. S2 and Fig. 4). When TAMRA-labeled anti-miR was combined with 1.5 equivalents of IgG-9r, approximately 16% of the anti-miR did not form a complex and remained as a distinct band on the gel. In contrast, when IgG-9r(9r) was used, no remaining anti-miR was detected, indicating complete complex formation. Furthermore, both IgG-9r and IgG-9r(9r) completely formed complexes with anti-miR when 4.0 equivalents were added. These findings indicate that the increased positive charges introduced by the arginine peptide in the anti-CD63 antibody enhanced its ability to form complexes with the negatively charged anti-miR.
The fluorescence intensity of TAMRA-anti-miR was quantified using ImageJ and the complex formation ability of ExomiR-Trackers were compared. Data are expressed as the mean ± S.D. The experiments were performed in triplicate.
Subsequently, we assessed the cellular uptake of ExomiR-Trackers in A549 cells. TAMRA-labeled anti-miR equipped ExomiR-Trackers were introduced into the culture medium of A549 cells. The cellular uptake of ExomiR-Tracker was analyzed using confocal laser scanning microscopy (Fig. 5). Notably, we observed a significant increase in fluorescence intensity when 4.0 equivalents of IgG-9r(9r) were used in the ExomiR-Tracker compared to the use of IgG-9r. We have already confirmed that arginine-modified anti-TSG101 antibody, which does not bind to the surface of exosome, is not taken up by cells,13) and that the uptake of ExomiR-Tracker is significantly lowered by treatment with GW 4869 which inhibit the production of exosome.13) These results suggest that Branch ExomiR-Tracker exhibits an enhanced capacity to deliver the loaded anti-miR intracellularly.
(A) Confocal imaging of ExomiR-Trackers equipped with TAMRA-anti-miR after 24 h of incubation. The final concentration of anti-miR and antibody was 150 and 600 nM, respectively. (B) Quantification of fluorescence intensity at 543 nm. Obtained images (n = 3) were analyzed by ImageJ and the fluorescence intensity was corrected by the number of cells. Data are expressed as the mean ± S.D. The experiments were performed in duplicate.
Finally, we investigated the functional inhibition of the target microRNA using ExomiR-Trackers through a dual luciferase reporter assay. As previously mentioned, miR-21 was selected as the target microRNA for ExomiR-Tracker. In this evaluation system, the expression of firefly luciferase had already been suppressed by endogenous miR-21. In this context, if the anti-miR-21 delivered by ExomiR-Tracker successfully inhibits target miR-21, it would expect a recovery in the luminescence intensity. Our findings revealed a significant recovery (approximately 50%) in luminescence intensity when Branch ExomiR-Tracker was used (Fig. 6 and Supplementary Fig. S3). Interestingly, the luminescence intensity in cells treated with Linear ExomiR-Tracker did not show any recovery. These results clearly demonstrate that Branch ExomiR-Tracker was efficiently introduced into A549 cells, and the anti-miR-21 component carried by Branch ExomiR-Tracker successfully inhibited miR-21 functions.
Evaluation of the inhibitory effects by ExomiR-Trackers on miR-21 functions by a dual luciferase reporter assay. The final concentration of anti-miR-21 was 150 nM and that of antibody was 600 or 900 nM. Data are expressed as the mean ± S.D. The experiments were performed in duplicate.
We modified ExomiR-Tracker by incorporating branched oligoarginine peptides and assessed its performance in terms of complex formation, cellular uptake, and gene expression inhibition in lung adenocarcinoma cells. The complex formation ability of IgG-9r(9r) was superior to that of the conventional IgG-9r. Furthermore, IgG-9r(9r) showed a remarkable capability to deliver anti-miR into lung adenocarcinoma cells compared to IgG-9r, leading to specific inhibition of the target miR-21’s function. These results can be attributed to the increased number of guanidino groups in arginine residues, which positively contribute to the loading efficiency and activity of anti-miR. In conclusion, Branch ExomiR-Tracker exhibits a promising potential as a tool for delivering nucleic acid drugs into lung cancer cells. The findings from this study offer novel insights into the exosome-hijacking DDS.
The antibody was treated with 20 equivalents of Traut’s Reagent in a 0.1 M phosphate buffer (pH 8.0) containing 2 mM ethylenediaminetetraacetic acid (EDTA). This reaction resulted in the formation of thiolated antibody (IgG-SH). Subsequently, IgG-SH was combined with 20 equivalents of a branched oligoarginine peptide ((D-Arg)9-Lys(D-Arg)9-Cys(Npys)-amide) in a 0.1 M phosphate buffer (pH 5.5). This process yielded the branched oligoarginine-introduced antibody (IgG-9r(9r)). To purify IgG-9r(9r), a Zeba Spin Desalting Column (MWCO = 40 k) was employed. The UV-vis absorbance of IgG-9r(9r) was measured using microvolume spectrophotometers (NanoDrop), enabling the determination of the antibody concentration.
Preparation of Branch ExomiR-Tracker (IgG-9r(9r)/Anti-microRNA)To develop Branch ExomiR-Tracker, purified IgG-9r(9r) and anti-miR were combined in PBS and incubated at 20 °C for 20 min. The complex formation ability of ExomiR-Tracker was assessed by subjecting the resulting ExomiR-Tracker to an EMSA.
Cellular Uptake of Anti-Exo and ExomiR-TrackerA549 cells were cultured on collagen-coated glass-bottom culture dishes at 37 °C in 5% CO2 for 24 h. Subsequently, the cells were supplemented with AF647-labeled antibodies (anti-CD63, anti-CD9, anti-CD81, and anti-TSG101) and incubated for an additional 24 h. Afterward, the cells were washed with PBS and fixed using a 4% paraformaldehyde-phosphate buffer solution. The cytoskeleton and nuclei were stained with phalloidin and Hoechst33342, respectively. Then, cellular uptake of antibodies was analyzed using confocal microscopy.
To investigate the exosome-dependent cellular uptake of antibodies, the same cell culture procedure as described above was followed for 24 h. The next day, the medium was replaced with Advanced-Dulbecco’s modified Eagle’s medium (DMEM), and the cells were supplemented with AF647-labeled antibodies with or without A549 exosome. After 12 h of incubation, the cells were fixed and stained using the aforementioned procedures.
For the evaluation of cellular uptake of ExomiR-Tracker, A549 cells were cultured on glass-bottom dishes for 24 h. Then, the cells were treated with ExomiR-Trackers containing TAMRA-labeled anti-miR. Following a 24-h incubation period, the cells were fixed, stained, and analyzed using the same aforementioned procedures.
Functional Inhibition of Branch ExomiR-TrackerA549 cells were cultured in D-MEM at 37 °C in 5% CO2. The cells were plated into 96-well plates (0.32 cm2/well) at a density of 5.0 × 103 cells/100 µL/well and cultured for 24 h. The following day, the cells were treated with ExomiR-Trackers for 24 h. After the incubation period, the medium was replaced with an antibiotic-free medium, and the cells were transfected with either pmiR-GLO plasmid (100 ng/well) or pmiR-21-reporter plasmid (100 ng/well) using Lipofectamine 2000, following the manufacturer’s instructions. After an additional 24 h of incubation, the cells were lysed, and the luciferase activity of the lysates was measured using a dual luciferase assay kit.
Materials and InstrumentsThe anti-CD63 antibody and anti-CD9 antibody were purchased from Cell Engineering Corporation (Osaka, Japan). Other antibodies, A549 exosome, act-stain488 phalloidin, and Fluoromount were purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). Luciferase assay-related reagents (pmiR-GLO plasmid, pmiR-21-reporter plasmid, passive lysis buffer, firefly luciferin, Renilla luciferin) were purchased from Promega (Madison, WI, U.S.A.). Furthermore, 4% paraformaldehyde was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). The glass-bottom culture dish was purchased from Matsunami Glass Ind., Ltd. (Osaka, Japan). Hoechst33342 was purchased from Dojindo Laboratories (Kumamoto, Japan). Cell culture-related reagents (D-MEM, penicillin/streptomycin solution, Trypsin, PBS) were purchased from FUJIFILM Wako (Osaka, Japan), and fetal bovine serum was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). The oligoarginine peptide (Cys(Npys)-(D-Arg)9-amide, (D-Arg)9-Lys(D-Arg)9-Cys(Npys)-amide) was synthesized by Peptide Institute, Inc. (Osaka, Japan), whereas the oligonucleotides (TAMRA-anti-miR, anti-miR-21) was synthesized by GeneDesign, Inc. (Osaka, Japan). All other reagents and materials were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.).
The cellular uptake of antibody and ExomiR-Trackers was analyzed using a confocal microscope (LSM800) (Carl Zeiss, Oberkochen, Germany). The EMSA was performed using the ChemiDoc Touch MP (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). The luminescence intensity was measured using a multimode plate reader (Cytation 3) (BioTek, Winooski, VT, U.S.A.).
This study was supported by the Grant-in-Aid for Transformative Research Areas (A) “Material Symbiosis” (Grant Number: 20H05874 awarded to A.Y. and T.Y.) from MEXT, Japan. This study was also supported by JSPS KAKENHI (Grant Numbers 22H00593 to A.Y. and 22J20228 to S.O.), Japan.
The authors declare no conflict of interest.
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