Abstract
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Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo
Abstract
Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosome-mediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells.
emerging evidence suggests that extracellular vesicles (EVs) are important vehicles mediating cell-cell cross talk and interorgan communications. Extracellular vesicles (EVs) are a group of heterogeneous, membrane-surrounded structures derived from a variety of cells, in the absence or presence of noxious stimuli. In current nomenclature, the term EV refers to exosomes (Exo), microvesicles (MV), and apoptotic bodies (AB), classified based on their sizes, components, and mechanisms of formation (36, 47). These evolutionally conserved vesicles exert essential physiological and pathological effects on both recipient and parent cells, probably via a variety of the important composition molecules that carry unique functions. Of the three different types of EVs, the majority of interests falls on the MVs and exosomes, given that ABs are most often derivatives of dying cells (47).
In recent years, strong interests center round the potentials of exosomes or MVs to serve as targets for the development of biomarkers or therapeutic reagents, particularly in the field of oncology (39, 41). Intensive studies on the role of EVs in cancer metastasis have shed light on using these cell type-specific vesicles to deliver cell-specific therapy against malignancy (27, 40). In noncancer diseases, EV-mediated cell-cell communications have also been reported (17, 22, 37). EV-containing miRNAs have been shown to play crucial functions in numerous devastating lung diseases and infections (14). In the pathogenesis of human diseases, the compositions of MVs and exosomes are robustly altered, including various miRNAs, suggesting the potential of using MVs or exosomes as for drug delivery. Exosomes are enriched in specific repertoires of miRNAs, rather than a mere reflection of the compositions of the “mother” cells. The profiles of exosomal miRNAs are specific to stimuli and cell types, reflecting the status of the “mother” cells (25, 43). Recent studies on the exosomal loading suggest that miRNAs are not randomly selected and loaded into exosomes. For example, hnRNPA2B1 has been shown to coordinate the sorting of a specific repertoire of miRNAs that share a consensus sequence into exosomes (44). RNA molecules that are enriched with 3′-UTR fragments are likely to be sorted into exosomes (2, 3). RNAs seems to be enriched in 3′-UTR fragments (2), which might be important for the sorting of specific mRNAs into these vesicles (3). Additionally, ESCRT machinery has also been reported to regulate the sorting of exosomal compositions into vesicles (31). Exosome maturation and release involve Rab proteins that regulate the intracellular vesicle budding, transport, tethering, and fusion. Furthermore, the role of the actin cytoskeleton in exosome release has been well reported (16). Invadopodia in cancer cells, actin-rich subcellular structure s, are known to control exosome secretion (16).
Currently, it is thought that EVs shuttle and protect EV-encapsulated miRNAs from enzymatic digestion and degradation (6, 13). Furthermore, self-produced EVs are unlikely to trigger immune responses in the host, given that they carry the same surface proteins as their parent cells. These features make EV-mediated small nucleotide delivery a promising system. Alvarez-Erviti et al. (1) have performed exosome-mediated siRNA delivery to the mouse brain. Host-derived dendritic cells are preengineered with the neuron-specific RVG peptide. Next, these dendritic cells are selected as donor cells for exosome production. In this study, electroporation is used to transfect exogenous siRNAs targeting housekeeping genes GAPDH or BACE1 (a therapeutic target for Alzheimer’s disease) into exosomes. However, the siRNA encapsulation efficiency is not addressed in their study (1). Nevertheless, after the manipulated RVG exosomes are injected intravenously in wild-type (WT) C57BL/6 mice, significant mRNA and protein knockdowns of BACE1 in the brain cortex are confirmed (1).
Despite promising results, significant obstacles exist on applying EV-mediated therapeutic agents. For instance, recent studies have shown that the number of copies of several “highly upregulated” miRNAs found in tumor cells are in fact very low in each individual exosome detected in plasma (7). Apparently, the host-derived exosomes are unable to deliver enough copies of the desired miRNAs without manipulation of their compositions. On the other hand, using circulating exosomes obtained from the host/patient serum probably will offer the best cell specificity and cause less allergic reactions or rejections by the recipients. There are several important issues that need to be addressed before the EV-mediated miRNA delivery can be used for drug development. For instances, how to enrich specific miRNA compositions in the EVs? Also, whether these EV-shuttling miRNAs indeed can have physiological or pathological effects on their recipient cells?
To address these questions, we explored a convenient and highly efficient method in which EVs can be directly transfected with miRNA mimics or inhibitors. To the best of our knowledge, this is a novel method using chemical based reagents to directly transfect miRNAs into host-derived EVs. This method is convenient and efficient and carries the potential to be used quickly for research and clinical applications. In this article, we compared this new method with electroporation-directed introduction of miRNAs into exosomes. Additionally, we investigated whether the exosome-mediated miRNA delivery is functional in the recipient cells. Given that exosomes contain less copies of miRNAs compared with microvesicles (10, 18), we decided to use exosomes in this report. However, similar methods can be adopted for MVs.
MATERIALS AND METHODS
Animal, cell culture, and isolation of alveolar macrophages.
Wild-type C57BL/6 mice of both genders (6 to 8 wk of age) were obtained from Jackson Laboratory. All the protocols involving animals in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Boston University.
RAW 264.7 cells [American Type Culture Collection (ATCC), Manassas, VA] were cultured in DMEM medium with 10% fetal bovine serum (FBS). Murine alveolar macrophage cell line MH-S and human monocyte cell line THP-1 (ATCC) were cultured RPMI-1640 Medium with 10% fetal bovine serum (FBS). The differentiation of THP-1 monocytes into macrophages was induced by 5 ng/mL PMA according to the previous report (35). All cells were cultured at 37°C in a humidified atmosphere of 5% CO2–95% air.
The method used for isolation of murine alveolar macrophages was descried previously (51). Briefly, after tracheostomy, 2 ml (1 ml × 2) of PBS were used to lavage total mouse lungs and bronchoalveolar lavage fluid (BALF) was obtained. BALF cells (typically >90% of these cells are macrophages) were collected after centrifugation at 400 g for 10 min.
Isolation and differentiation of bone marrow-derived macrophage.
Mouse bone marrow was isolated as previously described (29) and was cultured with 30% L929-cell-conditioned medium in DMEM complete medium for 7 days before any further experimental procedure. L929 cells were purchased from ATCC. To prepare L929-cell conditioned medium, L929 cells were cultured in DMEM media with 10% FBS at 37°C in a 5% CO2 incubator. Cell culture media were collected and filtered using 0.22-μm filters.
Cloning, transfection, and dual-luciferase reporter assay.
The fragment of human BCL-2 3′-UTR was amplified from cDNA using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA) and inserted into pRL-TK vector (Promega, Madison, WI) as described previously (49). The primers for human BCL-2 3′-UTR are as the following: forward primer (5′-TCTTCCTGAAATGCAGTGGTGCTTA-3′); reverse primer (5′-TTAGAGCAAGTGCAGCCACAATACT-3′). Reporter plasmid transfection into THP-1 cells was performed using Lipofectamine LTX (Invitrogen, Waltham, MA) according to the manufacturer’s protocol. miRNA mimic control, inhibitor control, miR-15a mimic, or miR-15a inhibitor (Sigma, St. Louis, MO) was transfected separately into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Dual-Luciferase assay was performed using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol, 24 h after transfection.
RNA preparation, reverse transcription, and quantitative real-time PCR.
miRNeasy Mini Kits (cat. no. 217004; Qiagen, Valencia, CA) were used for purification of total RNA from cells. Exosomal RNA was isolated using Total Exosome RNA & Protein Isolation Kit (cat. no. 4478545; Thermo Fisher Scientific, Waltham, MA). Single-stranded cDNA was generated according to the manuals of the High-Capacity cDNA Reverse Transcription Kit (cat. no.4374966; Thermo Fisher Scientific). For miR-15a detection, real-time PCR was performed using TaqMan PCR Kit (cat. no. 4427975; Thermo Fisher Scientific) and Applied Biosystems StepOnePlus Real-Time PCR Systems. Human HPRT1 (cat. no. 4331182-Hs99999909_m1) or mouse Hprt (cat. no. 4331182-Mm03024075_m1) was used as normalization control, respectively.
Preparation of miR-15a mimic/inhibitor loaded-exosomes.
To introduce miR-15a mimic or inhibitor into exosomes, a modified method of calcium chloride transfection was developed. For a 60-mm dish (50–70% confluency) with 5 ml of exosome-free media, 200 pmol miRNA mimic or inhibitor were mixed with 20 μg exosomes in PBS, and then CaCl2 (final concentration 0.1 M) was added. The final volume was adjusted to 300 μl using sterile PBS. The mixture was placed on ice for 30 min. After being heat shocked at 42°C for 60 s, the mixture was placed on ice for additional 5 min. For the RNase treatment, exosomes were incubated with 5 μg/ml of RNase (EN0531; Thermo Fisher) for 30 min at 37°C as described before (20).
For electroporation, miRNA mimic or inhibitor was mixed with exosomes derived from the THP-1, RAW 264.7, MH-S, bone marrow-derived macrophage (BMDM), or BALF in a 1:1 (wt/wt) ratio in the electroporation buffer as described previously (12). The mixture was loaded into the Neon Tip and electroporated at 0.5 kV using 10-ms pulse five times using the Neon Transfection System, according to the manufacturer’s protocol (Thermo Fisher Scientific).
Exosome isolation and labeling.
Exosome-depleted FBS (EXO-FBS-250A-1; System Biosciences, Palo Alto, CA) was used for cell culture before the culture medium was collected. Exosomes were isolated using differential centrifugation and microfiltration, as previously described (23). Cells were removed after centrifuging at 500 g for 10 min. Supernatants were harvested and centrifuged at 2,000 g for 10 min to eliminate cells debris. The supernatant was then filtered through a 0.22-μm filter (EMD Millipore, Darmstadt, Germany). Next, the exosomes were pelleted after the ultracentrifugation at 100,000 g for 120 min at 4°C. PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma) were purchased and used according to manufacturer’s protocol.
Western blot analysis.
Western blot analysis was performed as described previously (48). Briefly, cells were homogenized in RIPA lysis buffer supplemented with Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Sigma). Protein lysates were resolved on SDS-PAGE gels before transferred to the PVDF membrane (EMD Millipore). Anti-BCL2 antibody was obtained from Cell Signaling Technology (Danvers, MA).
Mouse monoclonal anti-GAPDH (Thermo Fisher Scientific) and anti-tubulin (Sigma) were used as a loading control. The densities of bands were quantitated using ImageJ software.
Dynamic light scattering and transmission electron microscopy.
Dynamic light scattering (DLS; Brookhaven 90plus Nano-particle Sizer) was performed to determine the average size of exosome. The exosome preparation kit for transmission electron microscopy imaging was obtained from 101Bio (Palo Alto, CA). The transmission electron microscopy (TEM) images were taken using a Philips CM120 EM.
Statistical analysis.
All data are presented as means ± SD. All the data from three independent experiments were averaged before normalization. For quantitative real-time PCR, the same amount of cDNAs were used and all the data were analyzed at the same time. For Western blotting, the pictures with similar exposure time were used for data analysis. Comparisons between two groups were performed using a two-tailed unpaired Student’s t-test. Multiple groups were compared using a one-way ANOVA with Tukey’s method. P < 0.05 was considered statistically significant.
RESULTS
Enhancement of specific miRNA or its analogs in mammalian cell-derived exosomes.
We illustrate a novel strategy to prepare the miRNA mimic or inhibitor-loaded exosomes as shown in Fig. 1A. Calcium chloride-associated transfection methods are widely used for introducing DNA into mammalian cells (19). We used the above method to introduce selected miRNAs into the isolated exosomes directly and modified the protocol with additional heat shock. In addition, we also used electroporation to directly introduce miRNAs into the isolated exosomes (Fig. 1B). The efficiency and convenience were compared between these two strategies.
Characterization of exosomes derived from THP-1 macrophages. A: flowchart illustrating the method to introduce microRNA (miRNA) mimics or inhibitors into exosomes via modified calcium chloride transfection. B: flowchart illustrating the method to introduce miRNA mimics or inhibitors into exosomes via electroporation. C: transmission electron microscopy (TEM) image of exosomes isolated from THP-1 cells (scale bar = 100 nm). D: expression of CD9, CD63, Sp1, and Flot-1 in THP-1 cell lysis and exosomes. Ten micrograms of protein were used for Western blot analysis. E: dynamic light scattering (DLS) analysis of exosomes isolated from THP-1 cells. This size distribution is displayed as a plot of the relative intensity of light scattered by particles (on the y-axis) vs. various size classes (on the x-axis) that are logarithmically spaced.
We first isolated exosomes derived from PMA-differentiated THP-1 cells (adherent macrophages) using ultracentrifugation, as described in materials and methods. The morphology and size were examined and confirmed using TEM and DLS, respectively (Fig. 1, C and D). Given the well-known difficulty of conveniently transfecting mammalian macrophages (50), we intentionally used macrophage-derived exosomes in this report to test our methods. The TEM image showed stained vesicles with typical exosomal morphology as reported previously (23) (Fig. 1C). Next, we used DLS to measure the size distribution of the exosomes derived from THP-1 macrophages. As shown in Fig. 1D, the size distribution of isolated vesicles was 86.5 ± 2.8 nm, which fell into the exosomal ranges, from 50 to 100 nm as described previously (45).
To determine the efficiency of loading methods, we purified the RNAs from the exosomes that were loaded with control mimics or miR-15a mimics. MiR-15a has been reported to play an important role in native immunity and host defense (29, 38). Therefore, we chose miR-15a as an example to test the efficiency of our method, given the convenience to analyze the functional status of exosome-mediated introduction of miR-15a in the following steps. Semiquantitative RT-PCR was performed to measure the amounts of exosomal miR-15a after calcium chloride-mediated transfection or after electroporation. The introduction of miR-15a was highly detected in the exosomes that were directly transfected using modified calcium chloride-mediated transfection with heat shock or using electroporation (Fig. 2A, lanes 2 and 6, from left to right). No detectable miR-15a was found in the exosomes that were transfected using the calcium chloride-mediated transfection alone or heat shock alone. Meanwhile, no accumulation of miR-15 in exosomes was observed in negative control groups (Fig. 2A, lanes 1, 3, 4, and 5, from left to right). Thus both modified calcium-mediated transfection plus heat shock and electroporation successfully introduced the miRNA molecules into the isolated exosomes directly.
In vitro delivery of miR-15a mimic or inhibitor-loaded exosomes to THP-1 macrophages. A: control or miR-15a mimics were introduced into exosomes as indicated. After being washed, RNA samples were isolated from purified exosomes. The expression of miR-15a in exosomes was detected using semiquantitative RT-PCR. U6 snRNA was used as an internal control. B: exosomes derived from THP-1 cells were labeled with PKH26 and added to the cell culture medium. The same labeling, washing process without exosomes, was used as negative control (no exosome). Images showing exosome uptake into THP-1 cells were captured at the indicated time points (scale bar = 100 μm). C: overexpression of miR-15a after addition of exosome-shuttling miR-15a mimics to the cell culture medium. Twenty-four hours after incubation, the expression of miR-15a in THP-1 cells was detected using real-time RT-PCR. D: inhibition of miR-15a after adding exosome-shuttling miR-15a inhibitors to the cell culture medium. Twenty-four hours after incubation, the expression of miR-15a in THP-1 cells was detected using real-time RT-PCR. E: overexpression of miR-15a using electroporation. MiR-15a was introduced into exosomes using electroporation. 24 h after addition of electroporation-treated exosomes, the expression of miR-15a in THP-1 cells was detected using real-time RT-PCR. F: inhibition of miR-15a using electroporation. MiR-15a was introduced into exosomes using electroporation. 24 h after addition of electroporation-treated exosomes, the expression of miR-15a in THP-1 cells was detected using real-time RT-PCR. All the data represent three independent experiments. *P < 0.05, **P < 0.01.
Determine the uptake of exosomes by macrophages.
Growing evidence has shown that exosomes can be internalized into the recipient cells (30). The most common method used for monitoring exosome uptake involves staining the exosomal membranes using fluorescent lipid membrane dyes, such as PKH67 (11), PKH26 (5), DiI (26), and DiD (15). In our studies, we used PKH26 to label the exosomes and monitored the uptake of labeled exosomes by the recipient cells. THP-1 macrophage uptake of the labeled exosomes was viewed under fluorescent microscope and is shown in Fig. 2B. Furthermore, the uptake of exosomes by THP-1 macrophages indicated a time-dependent manner (Fig. 2B).
Quantify the efficiency of exosome-mediated miRNA delivery in vitro.
Next, we evaluated the efficiency of exosome-mediated miRNA delivery in vitro. For the modified calcium-mediated transfection, we first added miR-15a mimic or inhibitor-loaded exosomes into the cell culture of THP-1 cells. The relative expression levels of miR-15a were detected using real-time PCR 24 h after incubation. Successful overexpression or suppression of the miR-15a level was confirmed in THP-1 macrophages that were treated with miR-15a mimic-enriched exosomes or miR-15a inhibitor-loaded exosomes, respectively (Fig. 2, C and D). This data demonstrated that the exosomes enriched with miR-15a mimics or inhibitors were internalized by macrophages successfully. Additionally, the exosome-enwrapped miRNAs were functional after uptake by the recipient cells. We also repeated the above experiments using the miR-15a mimic or inhibitor-enriched exosomes that were generated via electroporation. The results were similar and are shown in Fig. 2, E and F. These results indicated that exosomes serve as an efficient vehicle to transport miRNA mimics or inhibitors to the recipient cells.
MiR-15a mimic or inhibitor-enriched exosomes are functional in THP-1 macrophage.
To determine whether miR-15a mimic or inhibitor-enriched exosomes are functional after being up taken by the recipient cells, in this case, the THP-1 macrophages, we analyzed the BCL-2 gene expression using the luciferase assays. BCL-2 was chosen as a read-out here given that it is a direct target gene of miR-15a reported previously by Cimmino et al. (8). The 3′-UTR of human BCL-2, which contains the miRNA response element for miR-15a, was cloned into a Renilla luciferase reporter (Fig. 3A). Significant changes in the luciferase activity in THP-1 macrophages were detected after introduction of the miR-15a mimic or inhibitor-enriched exosomes. In these exosomes, miR-15a mimics or inhibitors were introduced using the modified calcium-mediated transfection (Fig. 3B). Similar results were observed using the exosomes that were directly transfected via electroporation (Fig. 3C). Moreover, using the Western blot analysis, we confirmed that BCL-2 protein levels were also altered in the THP-1 cells after being exposed to the miRNA-loaded exosomes. As shown in Fig. 3, D and E, exosome-shuttling miR-15a mimics or inhibitors introduced using the either of the two distinct methods effectively altered the BCL-2 expression levels.
MiR-15a mimics or inhibitors delivered via exosomes were functional in THP-1 macrophages. A: miR-15a regulatory element in BCL-2 3′-UTR is shown. Sequence alignment of miR-15a and the BCL-2 3′-UTR from different species is also shown. B and C: MiR-15a mimics or inhibitors were introduced into exosomes using modified calcium chloride transfection (B) or electroporation (C). Firefly luciferase reporter and Renilla luciferase reporter containing BCL-2 3′-UTR sequences were cotransfected into THP-1 cells. Next, miR-15a mimic or inhibitor-loaded exosomes were added to the culture medium. Renilla luciferase activity was measured and normalized to firefly luciferase activity 24 h after transfection. D–G: miR-15a mimics or inhibitors were introduced into exosomes using modified calcium Chloride transfection (D and E) and electroporation (G and H). Western blot analysis of BCL-2 was performed using the protein lysates obtained from THP-1 cells that were incubated with miR-15a mimic or inhibitor-loaded exosomes for 24 h. Protein levels were quantified using densitometry and normalized to tubulin (E and H). Results represent the average values obtained three independent experiments. RLU, relative light units. *P < 0.05, **P < 0.01, ***P < 0.001.
Introduction of miR-15a molecules via exosomes into various murine macrophages in vitro.
To further confirm our observations, we repeated these experiments using three different murine macrophages in addition to human THP-1. First, we prepared three types of exosomes derived from RAW 264.7 cells, MH-S cells, and BMDM, respectively, using the methods detailed in materials and methods. MiR-15a mimics or inhibitors were loaded into the exosomes using modified calcium-mediated transfections. The miR-enriched exosomes were added into cell cultures of macrophages. The uptake of miR-enriched exosomes into the RAW 264.7 cells (Fig. 4A), MH-S cells (Fig. 4D), and BMDM (Fig. 4G) was visualized 24 h after incubation. The effects of overexpression or suppression of miR-15a were confirmed in RAW 264.7 cells (Fig. 4B), MH-S cells (Fig. 4E), and BMDM (Fig. 4H). Consistent with the data obtained using real-time PCR, the protein levels of Bcl-2 were either elevated or reduced in the three types of murine macrophages, respectively (Fig. 4, C, F, and I).
MiR-15a mimic or inhibitor-loaded exosomes generated using the modified calcium chloride transfection were functional in a variety of murine macrophage lines. Three different types of macrophage cells, RAW 264.7 (A–D), NH-S (E–H), or bone marrow-derived macrophage (BMDM) cells (I–L) are used, respectively, in this experiment. Exosomes derived from RAW 264.7 cells, MH-S, or BMDM cells were enhanced with miR-15a mimics or inhibitors using the modified calcium chloride-mediated transfection and then labeled with PKH26. The same labeling, washing process without exosomes was used as negative control (no exosome). Exosomes being up taken into the recipient cells were captured in images 24 h after adding exosomes to the cell culture medium (scale bar = 100 μm) (A, E, and I). After the treatment of miR-15a-enriched exosomes, the level of miR-15a in the recipient cells is shown in B, F, and J, respectively, detected using real-time RT-PCR. The protein level of Bcl-2 was determined using Western blot analysis (C, G, and K). Protein levels were quantified using densitometry and normalized to tubulin or GAPDH (D, H, and L). All the data represent 3 independent experiments. *P < 0.05, **P < 0.01.
We also used the electroporation to introduce miR-15a mimics or inhibitors into the exosomes isolated from the above mentioned three types of macrophages. As expected, exosomes taken up by RAW 264.7 cells (Fig. 5A), MH-S cells (Fig. 5D), and BMDM (Fig. 5G) were confirmed. Consistently, the levels of miR-15a (Fig. 5, B, E, and H) and Bcl-2 (Fig. 5, C, F, and I) were robustly altered in these macrophages accordingly.
MiR-15a mimic or inhibitor-loaded exosomes generated using the electroporation were functional in a variety of murine macrophage lines. We again used the same 3 types of macrophage cells, RAW 264.7 (A–D), MH-S (E–H), or BMDM cells (I–L) in this experiment. Exosomes derived from RAW 264.7 cells, MH-S, or BMDM cells were enhanced with miR-15a mimics or inhibitors using electroporation and then labeled with PKH26. The same labeling, washing process without exosomes was used as negative control (no exosome). Exosomes being up taken into the recipient cells were captured in images 24 h after addition of exosomes to the cell culture medium (scale bar = 100 μm) (A, E, and I). After the treatment of miR-15a-enriched exosomes, the level of miR-15a in the recipient cells is shown in B, F, and J, respectively, detected using real-time RT-PCR. The protein level of Bcl-2 was determined using Western blot analysis (C, G, and K). Protein levels were quantified using densitometry and normalized to tubulin or GAPDH (D, H, and L). Results represent the average values obtained 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Delivery of miR-15a mimic or inhibitor-loaded exosomes to alveolar macrophages in vivo.
Our ultimate goal is to use exosomes as a vessel to deliver miRNAs in vivo and develop novel therapeutic strategies. Therefore, it is crucial to evaluate whether miR-enriched exosomes are effectively delivered to target cells in vivo. Here we used the exosomes that were isolated from mouse BALF as vehicles to deliver miRNA mimics or inhibitors to the lung. MiR-15a mimics or inhibitors were transfected into the exosomes using the above-mentioned modified calcium-mediated transfection. The loaded exosomes were then administered to wild-type C57BL/6 mice via inhalation. BALF was obtained 24 h after inhalation of exosomes. The uptake of the exosomes by alveolar macrophages was confirmed in the images captured using florescent microscopy (Fig. 6A). Moreover, the levels of miR-15a in alveolar macrophages were markedly altered after inhalation of miR-enriched exosomes, consistent with the results obtained using macrophages in vitro (Fig. 6B). We repeated the experiments using the exosomes transfected via electroporation. Consistent results were obtained. We confirmed that miRNA mimics or inhibitors were effectively delivered to the recipient alveolar macrophages via exosomes in vivo (Fig. 6, C and D).
Delivery of miR-15a mimic or inhibitor-loaded exosomes to the alveolar macrophage in vivo. A: exosomes derived from BALF were transfected with miR-15a mimic or inhibitor using modified calcium phosphate transfection and then labeled with PKH26. Twenty-four hours after intra-tracheal delivery of exosomes in mice (n = 4 for each group), exosomes being taken up into the alveolar macrophages were imaged. A representative image is shown in A (scale bar = 100 μm). B: the level of miR-15a in the recipient alveolar macrophages is shown in B, detected using real-time RT-PCR. C: exosomes derived from BALF were transfected with miR-15a mimic or inhibitor using electroporation and then labeled with PKH26. Twenty-four hours after intratracheal delivery of exosomes in mice (n = 4 for each group), exosomes being taken up into the alveolar macrophages were imaged. Representative image is shown in C (scale bar = 100 μm). D: the level of miR-15a in the recipient alveolar macrophages is shown in D, detected using real-time RT-PCR. Results represent the average values obtained 3 independent experiments. *P < 0.05, **P < 0.01.
DISCUSSION
miRNAs have an extensive role in human diseases and developing miRNA-based therapies has triggered strong interests. miRNAs, despite being promising therapeutic targets, often have multiple target genes. The potential of target effects may limit their applications in treating human diseases. Additionally, although miRNAs are relatively stable when compared with other RNA molecules, they remain vulnerable to intracellular and extracellular RNase-mediated digestion. Additionally, achieving relatively concentrated amounts of miRNAs in each EV and each recipient cell remains an unsolved issue. Recently, delivery of EVs loaded with miRNA analogs triggered extensive interests (42), A targeted, cell-specific delivery could minimize the side-effects of miRNAs, enhance their concentration per recipient cell, as well as limit the RNase digestion by encapsulating miRNAs into the EVs (28, 34). However, the strategies to make EVs suitable for the task of miRNA delivery remains incompletely explored. Previously, exosomes have been used to facilitate the delivery of siRNAs but not the miRNAs against the protein BACE1 for treatment of Alzheimer's disease (1). Furthermore, investigators have shown that there are only a small number of copies of the most prevalent miRNAs in each exosome (7). This finding further obscures the potentials of exosome-shuttling miRNA delivery.
To achieve the goal of using exosomes as a vehicle to better deliver the designated miRNAs, one needs first to develop a novel method by which miRNAs can be modulated in EVs directly, efficiently, and conveniently. Apparently, due the complex mechanisms of exosomal loading involving the ESCRT/Rab protein family, multivesicular bodies (MVBs), intracellular tubular, and actin networks, it is unreliable and unpredictable to generate selected miRNA molecule-loaded EVs via transfecting their mother cells.
Our method presented here has three significances: First, we reported a convenient and efficient method to enrich designated miRNA mimics or inhibitors in exosomes directly. Despite the success of using electroporation to enhance miRNA compositions in exosomes (28), electroporation does require special instrument and cannot be used in any laboratories who do not have this apparatus. This manuscript is intended to provide an easy, convenient, and reliable method.
Second, we demonstrated a novel method to transfect the difficult-to-transfect mammalian cells, such as primary cells or macrophages, using an exosome-based delivery. Third, we tested the probability to deliver interested miRNA molecules in vivo using exosomes as a vehicle. Liposomes have been previously used; however, they have poor uptake in some tissues and have significant toxic side-effects in many cells (4). Additionally, whether liposomal transfection can be used to safely deliver microRNAs in vivo remains unclear. Exosomes are generated by host cells and are nonimmunogenic in nature due to similar composition as their “mother” cells. Furthermore, tissue-specific delivery using cell-specific exosomes is currently under investigation by many groups. Given all the above-mentioned advantages, exosome-mediated delivery holds promising potentials in the near future, compared with the liposomal manner.
Although EVs contain MVs, ABs, and/or exosomes, in our studies, we chose to use exosomes for the following reasons. First, each exosome has been reported to carry less copy numbers of miRNAs (10, 18). The sizes of exosomes are smaller. Therefore, it makes the exosome a potentially practical vehicle to deliver miRNAs via inhalation in vivo, particularly to the lung parenchyma. Depending on their sizes, inhaled particles deposit in different areas of the lung. Generally, a particle with a size <1 µm is prone to reach the lung alveoli (33). Particles between 1 and 5 µm tend to deposit in the large airways or oropharynx (33). Exosomes typically are 50 to 100 nm in size and MVs are slightly larger (45, 47). ABs normally refer to the vesicles derived from dying cells and range from 1 to 5 µm (47). Therefore, exosomes and MVs fall into the appropriate sizes to serve as a vehicle to deliver miRNA molecules and drug molecules to lung alveoli and/or distant airways. Given the advantages of their particle sizes, growing interests have emerged to use exosomes as a vehicle to deliver specific miRNAs in vitro and in vivo (28, 34). However, the lack of efficient and convenient methods to enhance interested miRNAs in exosomes remains one of the biggest obstacles. In this report, we attempted to develop a novel method by which the specific miRNAs can be manipulated efficiently via direct transfection of miRNA analogs into exosomes. Here we validated this protocol using macrophage-derived exosomes as mentioned above. We chose miR-15a to test our strategies given that miR-15a is an essential regulator in native immunity and host defense (29, 38). It is convenient to test its functional status after reaching the recipient cells/tissue/organs. We believe that this method can be easily applied to all other different miRNA mimics or inhibitors as well.
The conventional calcium chloride-mediated transfection method is to introduce DNAs into cells. It is based on the formation of a calcium chloride-DNA precipitate. Calcium chloride (CaCl2) assists the interactions between DNA molecules and the cell surface, followed by endocytosis of the DNA molecules (46). We modified this protocol to facilitate the introduction of miRNA molecules into the exosomes. Although CaCl2 probably can form a complex with miRNA molecules, we anticipated that exosomes would fail to uptake the CaCl2-miRNA complex without heat shock (Fig. 2A, lane 5).5). Thus we modified the protocol by adding a step of heat shock. Exosomes, smaller than most bacteria, probably have no capacity to engulf any molecules via endocytosis. However, the exosomal membrane was derived from cellular membranous system and carried the same surface proteins and characteristics (47). Heat shock changed the fluidity of the plasma membrane (24), which is crucial for the entrance of DNA or RNA molecules into the cells. Therefore, it is expected that heat shock can also change the fluidity of exosomal membrane.
Furthermore, we compared the CaCl2-heat shock method with the previously used electroporation. A similar efficiency of miRNA introduction was observed. Electroporation, also referred as electropermeabilization, has been widely used in the transformation of DNA and miRNA molecules into the bacterial cells and the transfection of genes into cultured mammalian cells (9, 21). The well-recognized mechanisms of electroporation include the creation of nanometer scale water-filled holes in the membrane. During the process of electroporation, the lipid molecules of plasma membrane shift positions and open up a pore that serves as the conductive pathway for outside molecules to pass through (32). Our success on introduction of miRNA molecules into exosomes using the conventional electroporation further confirmed that the membrane structure surrounding exosomes carried the same characteristics as the plasma membrane of mammalian cells or bacterial cells. Our method has similar efficacy of transfection when compared with electroporation but does not require specific laboratory apparatus.
We confirmed the overexpression or suppression of miRNAs in the recipient cells after exposure to miRNA-transfected exosomes, both in vitro and in vivo. Moreover, the exosome-shuttling miRNAs are functional, as indicated by the alterations of their target genes in the recipient cells, such as Bcl-2 (Fig. 3, D and E). This result is encouraging and potentially sheds a light on future drug delivery vehicles, particularly those for cell-specific drug delivery and in vivo delivery. That being said, our targeting recipient cells were macrophages. Macrophages may uptake a majority of the delivered exosomes via phagocytosis. Therefore, whether exosome-mediated miRNA delivery can be cell-specific requires further investigation using other cell types, such as endothelial cells or epithelial cells.
Taken together, growing interests have emerged recently to use exosomes as a cargo to deliver specific miRNAs in vitro and in vivo. We developed a method by which the specific miRNAs in exosomes can be manipulated efficiently via direct transfection of miRNA analogs into the exosomes (Fig. 7). Our findings may provide a useful technical advancement in the development of exosome-mediated drug delivery.
Strategy to deliver miRNA-loaded exosomes in vivo and in vitro. Schematic experimental design for delivering miRNA-loaded exosomes.
GRANTS
This work was supported by the National Institutes of Health Grants R01-HL-102076, R21-AI-121644, and R01-GM-111313 (all to Y. Jin).
AUTHOR CONTRIBUTIONS
D.Z. and H.L. performed experiments; D.Z. and H.L. analyzed data; D.Z. and H.L. interpreted results of experiments; D.Z., H.L., and Z.Z. prepared figures; D.Z., H.L., Z.Z., J.K.M., and Y.J. drafted manuscript; D.Z., Z.Z., J.K.M., and Y.J. edited and revised manuscript; H.L. conceived and designed research; Y.J. approved final version of manuscript.
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Hui Chen and Dr. Joel Henderson at Experimental Pathology Laboratory Service Core of Boston University School of Medicine for the help with TEM. We also thank Dr. Xin Brown for the use of DLS at BioInterface Technologies (BIT) Facility of Boston University College of Engineering.
REFERENCES
Articles from American Journal of Physiology - Lung Cellular and Molecular Physiology are provided here courtesy of American Physiological Society
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Funding
Funders who supported this work.
HHS | National Institutes of Health (NIH) (3)
Grant ID: R01HL102076
Grant ID: R21AI121644
Grant ID: R01GM111313
NHLBI NIH HHS (1)
Grant ID: R01 HL102076
NIAID NIH HHS (1)
Grant ID: R21 AI121644
NIGMS NIH HHS (1)
Grant ID: R01 GM111313