Improvement of stem cell-derived exosome release efficiency by surface modified nanoparticles

doi:10.21203/rs.3.rs-42143/v1

Background: Mesenchymal stem cells (MSCs) are pluripotent stromal cells that release extracellular vesicles (EVs). EVs contain various growth factors and antioxidants that can positively affect the surrounding cells. Nanoscale MSC-derived EVs, such as exosomes, have been developed as bio-stable nano-type materials. However, some issues such as low yield and difficulty in quantification limit their use. We hypothesized that enhancing exosome production using nanoparticles would stimulate intracellular molecules.

Results: Our aim in this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified, positively charged nanoparticles and exosome generation from MSCs. We determined that Rab7, which is located in the multiple vesicle body and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation.

Conclusions: Nanoparticles may migrate to lysosomes during treatment. Simultaneously, intracellular exosome-forming factors can be stimulated during endosomal maturation. MSC-derived exosomes using nanoparticles may increase exosome yield and enable the discovery of nanoparticle-induced genetic factors.

Nanoscience

Autophagy

Mesenchymal stem cell-derived exosome

Positively charged nanoparticle

PLGA-PEI NPs

Rab7

Therapeutic clinical studies using mesenchymal stem cells (MSCs) have been reported recently, and techniques using MSC-derived exosomes are being actively developed. Several studies have explored the application of cell-derived exosomes using medical approaches. MSC-derived exosomes have been used to suppress immune responses or as a therapeutic treatment for cancer [1, 2]. In one approach, biocompatible materials are used to increase the quantity of exosomes in cell-free therapies [3, 4]. However, protocols have not been established to obtain a consistent yield and an efficient exosome extraction process for medical use.

The yield of exosomes derived from stem cells is very low, which warrants optimization of conditions to increase yields. Sucrose gradient-based ultra-centrifugation is commonly used to obtain material for various applications [5–7]. Extracellular vesicles (EVs) are important in cell-to-cell communication through the delivery of biological content, including intercellular proteins, lipids, and nucleic acids. Nanoscale EVs are difficult to maintain, and exosomes originating from the multiple vesicle body (MVB) are difficult to quantify [8, 9]. Quantification of the released exosomes is generally done by quantifying protein or microRNA (miRNA), although no formal method has been established [10, 11]. Exosomes are currently quantified by measuring the amount of total protein expressed in the exosome membrane or by confirming the expression of specific proteins, such as CD81, CD9, or CD63 [12, 13]. Techniques to extract exosomes and predict their quantity using absorbance values have been reported using cytokine assays or enzyme-linked immunosorbent assay analysis [2].

In this study, bioavailable nanoparticles (NPs) based on iron oxide with poly (lactic-co-glycolic acid), or PLGA, were used to improve the yield and expression of useful factors. We investigated the effects of NP surface charge and external stimuli on the generation of exosomes, with the goal of amplifying the yield of exosomes for applied research [14, 15]. The unique antioxidant and growth factors contained in exosomes were also considered. Iron oxide-based superparamagnetic iron oxide NPs (SPIONs) are bio-stable particles that have been approved by the United States Food and Drug Administration [16, 17]. NPs encapsulated with PLGA and polyethyleneimine (PEI) are approximately 100 nm in size and are used for drug delivery and clinical applications [15, 18, 19]. Altering the surface charge of NPs has demonstrated that positively charged NPs are more readily introduced into cells than are negatively charged NPs [16, 20]. The reason for using magnetic iron oxide NPs in this study was to introduce more NPs into cells using magnetic force. A clinical study reported using magnetism to deliver drugs into cells [19]. We utilized such iron oxide-based NPs to develop a technique to improve the production of exosomes from stem cells. We analyzed the mechanisms between NPs and the generation of exosomes and discovered an indicator related to exosome release by stimulating MVB formulation. These results will inform improvements in the composition and yield of exosomes.

Synthesis and characterization of polymeric clustered superparamagnetic (PCS) NPs

Magnetic nanoparticle solution (SPIO)-based polymeric clustered superparamagnetic (PCS) NPs were prepared with different surface modifications [21]. Modifying NPs with silica or other components cannot take advantage of their physical capabilities. However, the use of SPIO has the advantage of introducing more NPs into cells [15, 22]. Surface charges vary depending on the composition of the NPs. In this study, SPIOs were composed using specific PLGA-to-PEI ratios (Additional file 1: Figure S1). This produced two PLGA PCS NPs (negatively charged) and PLGA-PEI (PLGA 8 : PEI 2; positively charged). The zeta (ζ)-potential of the PLGA NPs was − 30 mV and that of the PLGA-PEI NPs was + 35 mV. Electron microscopy revealed that both NP types were approximately 100 nm in size (Fig. 1, Additional file 1: Figure S2). The surface properties of the NPs were also analyzed. NPs were stable in the medium and phosphate-buffered saline (Fig. 1f). Detailed properties and explanations are provided in the supporting information (Additional file 1: Figure S2).

Cellular uptake of PLGA-PEI PCS NPs and exosome release

To demonstrate the treatment conditions of PLGA-PEI PCS NPs (+) on MSCs, we measured the viability of MSCs using the CCK-8 assay after NP treatment. When the cells were exposed to different concentrations of PLGA-PEI PCS NPs (+) for 24 h, cell viability was 90.9 ± 8.14% with 5 μg/mL treatment (Fig. 2a). Higher concentrations of NPs decreased cell viability (73.6%). Cell viability was the most stable at 24 h using an NP concentration of 5 μg/mL (90.7 ± 4.1%) (Fig. 2b). When 10 μg/mL and 20 μg/mL NPs were used to treat cells, viability decreased to 86.2% after 6 h and to 86% after 1 h, respectively. The findings indicated that the concentration of 5 μg/mL was safe for internalization and that higher concentrations of positively charged PCS NPs were increasingly internalized by MSCs (Fig. 2c).

To determine the location of the NPs, MSCs were treated with 5, 10, and 20 μg/mL PCS NPs for 24 h and then imaged using transmission electron microscopy (TEM). The amount of NPs internalized increased depending on the concentration. The NPs appeared to be internalized in clusters rather than as single particles (Fig. 2d). The internalization of NPs was different depending on their surface charge. Positively charged PCS NPs tended to aggregate for internalization, whereas negatively charged NPs tended to be singular (Additional file 1: Figure S3).

Interestingly, PLGA-PEI PCS NPs were internalized into intracellular organelles for 24 h, which was thought to greatly influence the formation of exosomes. In Fig. 2d, the blue arrow denotes NPs and the red arrow denotes exosome and macrovesicles expression from MSCs. The quantity of exosomes increased proportionally depending on the concentration of PCS NPs (Fig. 2d). The size of exosomes ranged from 91 to 147 nm (Additional file 1: Figure S4).

To define the causes of exosome expression, we used two types of FluidMAG NPs (positive and negative) as controls. Exosomes were detected only in cells treated with positively charged NPs (Additional file 1: Figure S3). More positively charged particles adhered to the cell surface (Additional file 1: Figure S5). Exosomes were mainly observed at concentrations of positively charged PCS NPs that induced apoptosis, with increased expression of exosome protein markers detected by immunofluorescent (Fig. 2e) and western blot (Fig. 2f, g) examinations. To examine the quality of exosomes, we next attempted to establish exosome extraction conditions through effective NP processing using miRNA analysis.

Profiling of functional miRNAs in exosomes

To identify the overexpressed components in the exosomes in MSCs after treatment with NPs, we analyzed miRNAs. Fig. 3a depicts the comparison of the levels of expressed miRNAs in exosomes as heatmaps with fold change values. Twenty-nine miRNAs that displayed a fold change >1.5 were selected. miRNA counts were confirmed in the control and 5 μg/mL, control and 20 μg/mL, and 5 and 20 μg/mL groups (Fig. 3b). We focused on the miRNAs with increased expression. Comparisons according to log 2 and volume were used to select five representative miRNAs. In MSCs exposed to control and 5 μg/mL NPs, mmu-miR-2137, mmu-miR-3473b, mmu-miR-3473e, mmu-miR-3960, and mmu-miR-5126 displayed the highest volume and fold change values (Fig. 3c). In MSCs exposed to control and 20 μg/mL NPs, mmu-miR-2137, mmu-miR-3473b, mmu-miR-3473e, mmu-miR-5126, and mmu-miR-455-3p displayed the highest volume and fold change values (Fig. 3d). We conducted the analysis using miRNA database and predicted the genes that each miRNA could target. In addition, the selected predicted exosomal miRNA was identified (Additional file 1: Figure S6) and compared with the exosome components previously reported using the exoCarta exosome database (Table 1). The details of the miRNA analysis are summarized in Additional file 1: Figure S6. Importantly, we confirmed that the exosomes generated by the PCS NPs contained host factors affecting antioxidant effects, cell differentiation, and cell proliferation.

In addition, antioxidant factors were predicted by the representative miRNA identified in the 20 μg/mL and 5 μg/mL treatments. Other factors involved the physiological development of exosomes and binding of vacuoles, or the prediction of most of the receptor-generating miRNAs. Most importantly, exosomal miRNAs isolated from MSCs were differentially expressed upon treatment with 5 and 20 μg/mL of the positively charged NPs. The findings indicated that specific antioxidant or tissue regeneration factors may be obtained from MSC-derived exosomes by exposing the MSCs to different concentrations of NPs (Table 1, Additional file 1: Figure S6).

Endocytic mechanism of PLGA-PEI PCS NPs on MSCs

The changes in the internalization mechanism of iron oxide-based NPs were also examined to determine the factors involved in exosome generation. The surface modification of NPs can affect the interaction with cellular molecules. Several studies have reported a link between cytotoxicity and intracellular capacity [20, 23]. The absorption process is important in the physicochemical properties of the core particles and the coating. Surface charge is also involved in stimulating molecules in the cells [24]. Properties and particle size are very important determinants of the internalization mechanism [25]. While clathrin-mediated endocytosis internalizes NPs that are 120 nm in size, caveolae-mediated endocytosis internalizes NPs that are 60 nm in size [25, 26]. Thus, we used two types of inhibitors (Dynasore and Pitstop2) to inhibit the endocytosis pathways.

The two inhibitors did not completely inhibit the internalization of the NPs. Approximately 60% of the NPs were internalized in the Dynasore treatment. However, internalization of 5 μg/mL NPs was not completely inhibited when MSCs were treated with Pitstop2. The findings indicated that the PLGA-PEI PCS NPs were not internalized by clathrin-mediated endocytosis, and the approximately 40% inhibition by Dynasore was related to receptor-mediated endocytosis involving another mechanism (Fig. 4a, b). In addition, microscopy examination of the internalization of PLGA-PEI PCS NPs in viable MECs (Additional file 1: Figure S7) revealed the inhibition of internalization of the NPs by both Dynasore and Pitstop2 (Additional file 1: Figure S8a, b). To observe the transport mechanism of PLGA-PEI PCS NPs in MSCs, we used cellular markers of organelles related to endocytosis pathways [27, 28]. Early endosome antigen 1 (EEA1), Rab7, and GM130 were detected after MSCs were treated with 5 μg/mL NPs for 15, 30, and 60 min, and 6 h. As shown in Fig. 4c, NPs started to merge with EEA1 at 15 min. Since the NPs basically follow the endocytosis pathway, their transportation could be initiated in early endosome. Cell internalization efficiency exceeded 80% after 30 min. Interestingly, the merging efficiency of Rab7, which is also a late endosome and auto-phagosome marker, increased significantly after 6 h. The NPs appeared to have migrated to the late endosome following endosome maturation, and then to the larger organelles. NPs are considered to be transported to MVBs after superimposed with auto-phagosomes [29]. NPs also appeared with GM130, suggesting that some NPs were transferred to the Golgi. Several soluble and secretory molecules in the Golgi are involved in exocytosis. The imaging findings suggested that they may be related to exocytosis because they overlap with NPs [9, 12]. Using Lyso-Tracker we observed that the PCS NPs were finally transported to lysosomes by 24 h (Fig. 4d). However, it was not shown that PCS NPs were transported to the lysosome within 30 or 60 min. It is possible that the NPs could be degraded by lysosomal enzymes, whose activity would begin 6 h after the overlap with Rab7.

Another fact that can be inferred through the transport mechanism of NPs is that Rab7 is involved in MVB and the autophagosome formation [29, 30]. Rab7 have been implicated in the functioning of the class C core vacuole-endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) tethering complexes and are converted systemically in endosomal maturation [31, 32]. Presently, immunofluorescence analysis revealed that Rab7 and PCS NPs displayed a very wide overlap of 6 h in the organelles (Fig. 4c). Thus, it was hypothesized that the transport of NPs could be inhibited by the inhibition of Rab7 [33]. Rab7 expression was difficult to identify during the total reaction time of 6 h, and the merging efficiency decreased even when the cell area value was divided by the intensity value (Fig. 4e, f, Additional file 1: Figure S9). Interestingly, TEM revealed that the NPs in the majority of cells were trapped in vesicles that appeared to be early endosomes (Additional file 1: Figure S10). It was also difficult to find traces of exosome expression on the cell surface even after a prolonged time. The findings from the investigation of the NP internalization mechanism with Rab7 support the hypothesis that a factor influences the expression of exosomes during their merger with the MVB or lysosome at the late endosome.

Expression of autophagy-related genes

To understand exosome release, we needed to focus on the formation of MVBs. The internalization pathway of PLGA-PEI PCS NPs involved in exosome release was revealed, and the mechanism through the expression and inhibition of Rab7 was identified. This provided important evidence that MVB formation, including Rab7, is located in late-endosomes and autophagosomes [30, 34]. We previously assessed whether autophagy is induced in MSCs by externally introduced NPs [35]. We tried to measure the degree of change with time on the basis of the expression of markers of the most relevant autophagy processes. The temporal expression of genes during exposure to NPs was determined using reverse transcription polymerase chain reaction (Fig. 5). Primers were designed to confirm the autophagy process (Additional file 1: Figure S11) [36-38]. The expression of these genes during 1, 3, 6, 24, and 48 h of exposure to NPs confirmed that autophagy occurred for a short time.

Beclin-1 is expressed in the first phase of autophagy. A rapid increase in the expression of Beclin-1 was evident after 1 h of NP exposure (Fig. 5a). The expression of Beclin-1 subsequently decreased over time but remained statistically significantly greater than the corresponding control sample at each time. When the control group was set to 1 h, the value increased by approximately 23 times, indicating that the expression was rapid, and that autophagy had begun. Microtubule-associated protein 1 light chain 3 (LC3) was examined next. LC3, which is generally converted from the alpha to beta form during the formation of the phagophore, was introduced into the membrane (Fig. 5b, c). When observed at different times, LC3 displayed similar values between 1 and 3 h. The expression of p62 was highest at 1 to 3 h, and gradually decreased thereafter (Fig. 5d). This pattern may reflect the influence of other mechanisms after autophagy.

ATG5 and STX17 are genes that code for membrane proteins that are expressed at the end of autophagy when the autophagosome is combined with the autolysosome. ATG5 expression was similar after 6 h and then decreased slowly (Fig. 5e). STX17 also increased after 6 h (Fig. 5f). There was little difference in these factors in the formation of the autophagosome and vesicle binding showed little difference. Thus, autophagy by PLGA-PEI PCS NPs induced degradation through the combination of the lysosome and the autophagosome. NPs introduced by endocytosis induced the formation of autophagosomes and were subsequently degraded after transport to the autolysosomes.

Alteration of the quantity of NPs in MSCs by magnetic attraction

The SPIONs used in this study had the physical advantage of the capability to transport more NPs into cells. This experiment was conducted with the aim of increasing the release of exosomes by transporting more NPs into cells. MSCs were exposed to 5 μg/mL NPs for 1, 6, and 24 h in the absence or presence of magnets. We prepared a magnet 60 mm in size, which represents an intensity of approximately 0.6 T [21]. In the absence of the magnet, fluorescence-activated cell sorting revealed a gradual increase in the internalization rate of NPs over 24 h (Fig. 5g). In the absence of the magnet, the average mean intensity of NPs in MSCs was 2559.25 at 1 h, 9304.75 at 6 h, and 12581.25 at 24 h without a magnet. Detailed analysis of the intensity of NPs in the presence of a magnetic field for 1, 6, and 24 h is provided in Additional file 1: Figure S12.

In the presence of a magnetic field, NPs were increasingly internalized or adsorbed, compared to exposure to PCS NPs for 24 h (Fig. 5h). In a novel observation, the combination of NPs and exosomes reduced the loss of exosomes. The findings indicated that the expression of exosomes could be increased using positively charged NPs. NPs obviously stimulated the production of exosomes (Fig. 5h). In addition, we devised a formula to infer the quantity of exosomes derived from cells using imaging analysis to determine the exosome production (Fig. 5i).

We sought to demonstrate the association between the generation of exosomes and the use of surface-modified NPs. Future studies of the mechanism by which exosomes are generated from stem cells by NPs are required, as treatment with NPs is thought to affect the composition and yield of exosomes. Exosomes consist of double lipid membranes that encase 40–100 nm sized soluble proteins and RNA, and are considered to be important for intercellular communication [8].

Presently, NPs developed through the combination of PLGA and PEI were used to increase the expression of exosomes in cells. The enhanced expression involved the positively charged surface charge and the biocompatible PLGA [15]. Exosomes were expressed even when using commercially available positively charged NPs, but the amount was minimal. Therefore, the observation of the increased expression of exosomes using NPs was important. The analysis of miRNAs that were increased within exosomes also confirmed the expression of antioxidants and factors related to differentiation.

The presently-developed NPs displayed different internalization pathways depending on the surface potential. We demonstrated that the mechanism of dynamin-dependent endocytosis of nanoparticles in cells depended on the positively charged surface of PCS NPs. We explored the mechanism of overexpression of exosomes (Fig. 6). Rab7 was overexpressed at 6 h by the treatment of NPs. The positively charged NPs promoted the production of MVB and amplified the generation of exosomes. Verification was provided by the observations of amplified Rab7 expression at 6 h even if the Rab7 protein was inhibited. Rab7 is a protein which is located in the late endosome, autophagosome, and MVB membranes. However, when it is amplified, the significantly increased MVBs form exosomes. We conclude that NPs can induce the overexpression of Rab7 in the late endosome, MVB, and autolysosome membranes.

There are also autophagy factors expressed when MSCs are treated with NPs. The expression of STX17, which is involved in autolysosome formation, was confirmed by the expression of Beclin-1 at 1 h. These results confirmed that the expression of exosomes was due to the amplification of autophagy by the NPs introduced into the endosome, which resulted in the increase of autophagosomes and the accumulation of MVB in the cells .

We utilized iron oxide NPs to take advantage of the magnetic attraction technology [19], which introduces NPs into cells by magnetism [17, 20]. We designed NPs with iron oxide to introduce more NPs and used PLGA and PEI to make it easier for NPs to bind to the cell membrane. NPs became more internalized or adsorbed in the presence of a magnetic field than if they were exposed for 24 h. A novel observation was that NPs and exosomes could be combined to develop more exosomes without loss.

The present findings might be valuable in increasing the expression of exosomes using positively charged NPs. Clearly, NPs stimulated the production of exosomes. In addition, we were able to design a formula that inferred the amount of exosomes derived from cells using imaging analysis to determine the amount of exosomes produced (see the Methods section). This formula could be applied to increase the yield of exosomes in the laboratory but would be difficult to apply to clinical studies. Therefore, this study may influence future research through the expression of MSC-derived exosomes using positively charged NPs. Because the components of exosomes inherently depend on the concentration of NPs, it is expected that exosomes can be extracted from stem cells and used for desired purposes, such as tissue regeneration or antioxidant efficacy.

Currently studies using stem cell-derived exosomes have been actively conducted. therefore, the research to improve and apply the exosomes yield is very important commercially. Through this study, we developed positively charged nanoparticles based on iron oxide and PLGA that can increase the incidence of exosomes (Fig. 6). The research on the internalization mechanism of the nanoparticles and the factors affecting to increase in exosome yield were studied. It would be expected to develop into a nanoparticle-based technology to increase yield of exosome releasing derived from MSCs in the future.

PLGA-PEI and PLGA-Cy5.5 materials

Poly (D,L-lactide-co-glycolide) (PLGA, 50:50, MW 38000-54,000; Sigma-Aldrich, St. Louis, MO, USA) and polyethylenimine (MW ~ 25,000, M_n ~ 10,000 (PEI, Sigma-Aldrich) were used to synthesize the PLGA-PEI copolymer. N,N’-Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were prepared as a linker between the PLGA and PEI. PLGA (330 mg) and 113.67 mg of PEI, 18.33 mg of DCC and 11 mg of NHS were dissolved in 100 mL of dimethylsulfoxide (DMSO) solution and incubated for 24 h. After the completion of this reaction, the reaction mixture was dialyzed (MW cutoff = 10,000) with deionized water to remove excess DCC, NHS, and PEI. After dialysis, the polymer solution was freeze dried to obtain the powdered sample.
To synthesize the PLGA-Cy5.5 fluorescence polymer, 330 mg of PLGA was dissolved in DMSO along with 60 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Fisher Scientific, Waltham, MA, USA), 132 mg of NHS, and 9.9 mg of Cy5.5 (Cy5.5, red; Lumiprobe Co., Hannover, Germany), and incubated for 24 h. After the completion of this reaction, the reaction mixture was extensively dialyzed (MW cutoff = 10,000) with deionized water to remove excess EDC, NHS, and Cy5.5. The resulting PLGA-Cy5.5 polymer was freeze dried to obtain the powdered sample [15].

Synthesis of polymeric clustered SPIO (PCS) NPs

To manufacture the positively charged surface-modified NPs (PLGA-PEI PCS NPs), 0.1 mg of the PLGA-PEI copolymer and 0.4 mg of PLGA-Cy5.5 were dissolved in 0.1 mL DMSO. Iron oxide (II, III) magnetic NPs solution (0.1 mL containing 5 mg/mL) was added drop-wise to 3 mL of deionized water. The vial was vortexed for 5 min followed by 3 min of sonication. The solution mixture was stirred at room temperature for 6 h. At the end of the process, the obtained NPs were further purified by ultra-centrifugation and stored at 4°C until further use.
PLGA and SPIO were used to manufacture the negatively charged, surface-modified NPs. EDC and NHS were prepared as a linker between the Cy5.5 fluorescence molecule that was synthesized for the detection of NPs in the cells. Specifically, these PCS NPs, otherwise known as PLGA-Cy5.5, were synthesized by EDC-NHS coupling. For this, 330 mg of PLGA was dissolved in DMSO along with 60 mg of EDC, 132 mg of NHS, and 9.9 mg of Cy5.5, and incubated for 24 h. After the completion of this reaction, the reaction mixture was extensively dialyzed (MW cutoff = 10,000) with deionized water to remove excess EDC, NHS and Cy5.5. The resulting PLGA-Cy5.5 particles were freeze dried to obtain the powdered samples. We used 100% PEI to create positively charged NPs, and 100% PLGA to make negatively charged NPs. In addition, it has been made positively charged NPs with the composition of PLGA:PEI in an 8:2 ratio.

Characterization of NPs

The PCS NPs with a polymeric shell consisting of 10 nm diameter SPIOs in the core were prepared using an oil-in-water emulsion method. In a typical experiment, 1 mg of PLGA-Cy5.5 was dissolved in acetonitrile at 25°C. SPIO (0.1 mL containing 5 mg/mL) was added drop-wise to 3 mL of deionized water. Thereafter, the vial was vortexed for 5 min followed by 3 min of sonication. The solution mixture was stirred at room temperature for 6 h. At the end of the process, the obtained NPs were further purified by ultra-centrifugation and stored at 4°C until further use. For comparison, we purchased positively charged (FluidMAG-Chitosan, cat. 4118-1; chemicell GmbH, Berlin, Germany) and negatively charged (FluidMAG-CMX, cat. 4106-1; chemicell GmbH) NPs 100 nm in size. The surface charge was reported to be + 50 mV for FluidMAG-Chitosan [14] and -14 mV for FluidMAG-CMX [30].

The size and surface zeta potential of the NPs were obtained by dynamic light scattering using a Zetasizer-ZS90 apparatus (Malvern Instruments, Malvern, UK). The morphology of the particles was characterized by scanning electron microscopy (SEM) using a JSM-7100F instrument (JEOL, Tokyo, Japan). The samples for SEM were prepared by adding a droplet of the NP suspension (2 μL) to a polished silicon wafer. After drying the droplet at room temperature for 4 h, the sample was coated with platinum and imaged using SEM. To further understand the internal SPIOs core, TEM measurements were performed. The samples for TEM were prepared by the drop casting method [15, 25] over a carbon grid.

Cell culture

MSCs (cat. MUBMX-01001) and green fluorescent protein (GFP)-labeled MSCs (cat. MUBMX-01101) were purchased from Cyagen (Santa Clara, CA, USA). The cells were cultured in MEM-alpha containing 10% fetal bovine serum and 1× antibiotics. The medium was changed every 2 days after washing with Dulbecco's phosphate-buffered saline in a humidified atmosphere of 5% CO₂.

Flow cytometry analysis

The GFP-labeled MSCs were prepared in 60 mm culture dishes (1×10⁵ cells/well). After PCS NPs were added to the cells for 24 h, the cells were washed twice with PBS. Thereafter, samples were analyzed by flow cytometry using a FACS Aria3 flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed using FACS Diva software.

Viability assay

Cell viability was measured by counting viable cells using by the EZ-3000 CCK kit (Dogen, Seoul, Korea) (CCK-8). The MSCs were seeded (5×10² cells/well) into 96-well plates and incubated at 37°C in 5% CO₂ for 24 h. The next day, the culture medium was replaced with 200 μL of fresh medium containing different concentrations of PCS NPs. After incubation, 20 μL of CCK-8 was added and the cells were protected from light. The absorbance values at 450 nm were measured with an enzyme-linked immunosorbent assay reader after 4 h incubation.

Fluorescence analysis

The MSCs and GFP-labeled MSCs were seeded (5×10³ cells/well) into 4-well cell culture plates. The culture medium was replaced with fresh medium after 24 h of incubation and NPs were added. The MSCs were washed twice with PBS after incubation with NPs, fixed in 10% paraformaldehyde, and stained with 4′,6-diamidino-2-phenylindole (DAPI). Even though the NPs labeled with Cy5.5 (red) were easy to detect in cells, the cellular organelles were stained with specific antibodies in the cells. The primary antibodies used were anti-EEA1 (ab2900; Abcam, Cambridge, MA, USA) for early endosomes, anti-Rab7 (Aab126712; Abcam) for late-endosomes, and anti-GM130 (ab169276; Abcam) for the Golgi apparatus. To localize the PCS NPs in cellular organelles, goat anti-rabbit IgG H&L (Alexa fluor 488, green) secondary antibody (ab150077; Abcam) was used. In addition, Lyso-Tracker green DND-26 (cat. L7526; Life Technology, Carlsbad, CA, USA) was used to track the final location of the NPs in the cells. All samples were observed by confocal microscopy (Carl Zeiss Microscopy GmbH, Jena, Germany) and images were analyzed using ZEN lite ver. 2.3 software.

Inhibitors

Two inhibitors for the internalization of NPs were used: Dynasore (ab120192, Abcam) and Pitstop2 (SML-1169, Sigma-Aldrich). The concentration of both was 40 μM/dish and they remained active within 3 h prior to NP treatment. Dynasore inhibits dynamin, which is an important factor in receptor-mediated endocytosis 51. Pitstop2 inhibits clathrin-mediated endocytosis in the receptor-mediated endocytosis pathway. Both inhibitors were added to MSCs for 30 min before treatment with positively charged PCS NPs. CID 1067700 (cat. 2184, Axon Medchem, Groningen, Netherlands) was purchased to inhibit Rab7 by competitive inhibition.

TEM

Cells were fixed in 2.5% glutaraldehyde for 2 h at 4ºC and the specimens were solidified on 2% agar. Samples were post fixed in 1% osmium tetroxide after being washed with 0.1 M cacodylate buffer. Dehydration was performed in a step-wise manner using 50% to 100% ethanol. The samples were embedded in Epon resin. Ultrathin sections were cut using an ultra-microtome and stained using uranyl acetate and lead citrate. TEM was performed at 63 kV.

Preparation of exosomes

The medium of cultured MSCs was obtained to isolate exosomes using Total Exosome Isolation reagent (4478359; Invitrogen, Carlsbad, CA, USA), strictly according to the manufacturer’s instructions. PBS was used to re-suspend the purified exosomes. Exosomes were kept either at -80°C for long-term preservation or at -20°C for short-term preservation. Exosomal proteins were isolated using lysis buffer containing 0.1% Triton-X100 in PBS with a protease inhibitor cocktail.

Real-time RT-PCR

To prepare mRNA samples a total of 10 µL, including 2 µL of mRNA and 8 µL of reverse transcriptase reagents, was used. The mixture included 1 µL of 10× enzyme mix, 2 µL of 5× enzyme reaction buffer, and 5 µL of nuclease-free water. According to the manufacturer’s protocol on cDNA synthesis kit (TOYOBO, Osaka, Japan), were incubated at 42°C for 60 min and deactivated at 95°C for 5 min. cDNA was diluted 1:10 with 90 µL nuclease-free water for miRNA real-time RT-PCR.

Real-time RT-PCR was performed using the sequence detection system 7900 (Applied Biosystems, Foster City, CA, USA). Samples were subjected to reverse transcription using the SYBR Select master mix (Applied Biosystems) following the manufacturer’s protocol. The following primers were used for sequencing: 18S rRNA, forward: 5’- CTA ACC CGT TGA ACC CCA TT - 3’ and reverse: 5’- CCA TCC AAT CGG TAC TAG CG - 3’; Beclin-1, forward: 5’ - GGA GCT GGA AGA TGT GGA AA - 3’ and reverse: 5’ - ACT CCA GCT GCC TTT TA - 3’; LC3-α, 5’- GCC TGT CCT GGA TAA GAC CA - 3’ and reverse: 5’- GGT TGA CCA GCA GGA AGA AG - 3’; LC3-β, 5’- CCG AGA CCT TCA AGC AG - 3’ and reverse: 5’- CTC CCC CTT GTA TCG CTC TAT - 3’; p62, 5’- GTC TTG GGG AAG GGT TCA AT - 3’ and reverse: 5’-GGG GGT CCA AAG ACT TCA AT - 3’; ATG5, 5’- ATA TGA AGG CAC ACC CCT GA - 3’ and reverse: 5’ - CAT CCT TGG ATG GAC AGT GTA G - 3’; STX17, 5’- GAT CCC AAC AGA CCT GGA GA- 3’ and reverse: 5’- GAT ATT GGA GCG CAG TTG CT - 3’. The total reaction volume of 10 µL included 1 µL cDNA, 5 µL of premix, 1 µL of 10 pmol forward and reverse primers, and 3 µL of nuclease-free water. Real-time PCR cycle conditions were pre-amplification at 95°C for 3 min, 40 cycles of 95°C for 10 sec, 60°C for 60 sec, and melting curve analysis. All real-time PCR assays were performed in duplicate. Normalization of mRNA expression level was calculated using 18S rRNA.

miRNA analysis in exosomes

RNA samples were obtained from the medium treated with 5 or 20 μg/mL PCS NP for 24 h. The concentration of the sample was confirmed through Quality Control analysis and was confirmed to be between 0.17 and 0.26 ng/μL. To check the purity and quantity of RNA, a nanodrop was used to measure the absorbance at 260 nm and 280 nm. All raw data were extracted automatically in the Affymetrix data extraction protocol using the provided Affymetrix GeneChip® Command Console® Software (AGCC). The CEL files were imported, miRNA levels were normalized using the RMA algorithm, the detection above background p-values were calculated for all data, and the results were exported using the Affymetrix® Power Tools (APT) Software.

Array data were filtered by species-specific annotated probes. The comparative analysis between test and control samples was carried out using fold change. All statistical testing and visualization of differentially expressed genes was conducted using R statistical language 3.3.3 (https://www.r-project.org/).

Magnetic attraction

MSCs were sub-cultured (2×10⁵ cells) and incubated in a 60 mm dish. The next day, the NPs were added and a magnet the same size at the plate was immediately placed under the floor of the plate. The reaction time was changed to 1, 6, and 24 h, and then the cells were washed with PBS. Cells were detached with trypsin and the mean intensity value was measured using FACS in a round vial. In addition, electron microscopy images were analyzed in accordance with the TEM test conditions.

Imaging analysis quantification of exosome amounts

Statistical analyses

Real-time PCR results were analyzed to compare autophagy mRNA levels, Mann-Whitney U test and receiver operating characteristic curve analysis were performed using SPSS version 24.0 (IBM, Armonk, NY, USA). All graphs were plotted with GraphPad PRISM version 5.0 (GraphPad Inc., La Jolla, CA, USA). The sensitivity and specificity of the statistically significant mRNAs were analyzed with GraphPad PRISM version 5.0. p-values < 0.05 were considered to be statistically significant.

DCC

N,N’-Dicyclohexylcarbodiimide; EVs:Extracellular vesicles; MSCs:Mesenchymal stem cells; MVB:Multiple vesicle body; NHS:N-hydroxysuccinimide; NPs:Nanoparticles; PCS:Polymeric clustered superparamagnetic; PEI:Polyethylenimine; PLGA:Poly-D,L-lactide-co-glycolide; SPIONs:Superparamagnetic iron oxide NPs

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

All data analyzed during this study are included in this published article (Additional files 1 and 2).

Competing interests

The authors declare no competing financial interests.

Funding

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI19C1334).

This research was supported by the Development of Short-term Aging Murine Model for Age-related Hearing Loss (2019-51-0149), funded by the Chong Kun Dang Pharmaceutical Corp.

Author Contributions

D.J.P. designed the synthesis of exosomes with nanoparticles. W.S.Y. and W.C.K. supervised the construction and physicochemical characterizations in nanoparticles. J.-E. P., S.H.L., S.H., and J.S.C. supervised the biological assays. J.K. and Y.J.S provided support for this study. All authors reviewed the manuscript.

Acknowledgments

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI19C1334).

Authors' information

¹Department of Otorhinolaryngology, Yonsei University Wonju College of Medicine, 20 Ilsan-ro, Wonju, Gangwon-do 26426, Wonju, South Korea. ²Research Institute of Hearing Enhancement, Yonsei University Wonju College of Medicine, Wonju, South Korea. ³Department of Biomedical Engineering, Yonsei University, Wonju, South Korea. ⁴Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea

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Table 1 List of predicted target mRNAs from miRNA

miRNA	Predicted target mRNA		Functions and characteristics	Database on ExoCerta
miRNA	Symbol	Full name	Functions and characteristics	Database on ExoCerta
mmu-miR-2137	Gpx4	Glutathione peroxidase 4	Plays a key role in protecting cells from oxidative damage by preventing membrane lipid peroxidation	o
	Gosr1	Golgi SNAP receptor complex member 1	Regulates intracellular reactive oxygen species levels via inhibition of p38 MAPK (MAPK11, MAPK12, MAPK13, and MAPK14)	o
	Rab4A	Ras-related protein Rab-4A	Protein transport. Plays a role in vesicular traffic. Mediates VEGFR2 endosomal trafficking to enhance VEGFR2 signaling	o
	Prrx1	paired related homeobox 1	Enhances DNA-binding activity, and induces genes by growth and differentiation factors	Unknown
	Pex26	peroxisomal biogenesis factor 26	Peroxisome biogenesis factor 1, also known as PEX1	Unknown
mmu-miR-3473b	Rspry1	ring finger and SPRY domain containing 1	Extracellular region and zinc ion binding	o
	Ephb2	Eph receptor B2	Encodes a member of the Eph receptor family of receptor tyrosine kinase transmembrane glycoproteins	o
	Cplx2	complexin 2	Refers to one of a small set of eukaryotic cytoplasmic neuronal proteins that binds to the SNARE protein complex (SNAREpin) with high affinity	o
	Il-7	Interleukin 7	IL-7 stimulates the differentiation of multipotent (pluripotent) hematopoietic stem cells into lymphoid progenitor cells	o
	Rab11fip1	RAB11 family interacting protein 1	Forms a targeting complex that recruits a group of proteins involved in membrane transport to organelles	o
mmu-miR-3473e	St6galnac6	ST6-N-acetylgalactosaminide alpha-2,6-sialyltransferase 6	Modifies ceramides on the cell surface to alter cell-cell or cell-extracellular matrix interactions	o
	Adcy1	adenylate cyclase 1	Regulates various cellular functions via activating protein kinase A-dependent phosphorylation	o
	Wnk2	Serine/threonine-protein kinase WNK2	Plays an important role in the regulation of electrolyte homeostasis, cell signaling, survival, and proliferation	o
	Sioa1l3	Signal-induced proliferation-associated 1 -like protein 3	Plays a critical role in epithelial cell morphogenesis, polarity, and adhesion and cytoskeletal organization in the lens	Unknown
mmu-miR-5126	Atp6v1c2	V-type proton ATPase subunit C 2	Subunit of the peripheral V1 complex of vacuolar ATPase	o
	CD4	CD4 molecules	Integral membrane glycoprotein that plays an essential role in the immune response and serves multiple functions in responses against both external and internal offenses	o
	Pax2	paired box 2	Has a critical role in the development of the urogenital tract, the eyes, and the central nervous system	Unknown
	Aire	Autoimmune regulator	Expressed by a distinct bone marrow-derived population; induces self-tolerance through a mechanism that does not require regulatory T-cells and is resistant to innate inflammatory stimuli	Unknown
mmu-miR-455-3p	Tti1	TELO2-interacting protein 1 homolog	Phosphorylated at Ser-823 by CK2 following growth factor deprivation, leading to its subsequent ubiquitination by the SCF (FBXO9) complex	o
	Hk3	hexokinase 3	HK3 functions to protect the cell against apoptosis. Overexpression of HK3 has resulted in increased ATP levels, decreased reactive oxygen species production, attenuated reduction in the mitochondrial membrane potential, and enhanced mitochondrial biogenesis	o
	Sar1a	secretion associated Ras-related GTPase 1A	Involved in membrane trafficking. It is a monomeric small GTPase found in COPI vesicles. It regulates the assembly and disassembly of COPII coats	o
	Fgf4	fibroblast growth factor 4	Possesses broad mitogenic and cell survival activities and is involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, and tissue repair	Unknown
	Pex5	peroxisomal biogenesis factor 5	Targeting sequence involved in the specific transport of molecules for oxidation inside the peroxisome	Unknown
mmu-miR-877-5p	Gpx1	glutathione peroxidase 1	Belongs to the glutathione peroxidase family, members of which catalyze the reduction of organic hydroperoxides and hydrogen peroxide by glutathione and, thereby, protects cells against oxidative damage	o
	Aak1	AP2 associated kinase 1	Regulates clathrin-mediated endocytosis by phosphorylating the AP2M1/mu2 subunit of the adaptor protein complex 2 (AP-2), which ensures high affinity binding of AP-2 to cargo membrane proteins during the initial stages of endocytosis	Unknown
	Wnk3	WNK lysine deficient protein kinase 3	Plays a role in the increase of cell survival in a caspase 3-dependent pathway as a positive regulator of the transcellular Ca2+ transport pathway	Unknown
	Rab26	RAB26, member RAS oncogene family	Important regulators of vesicular fusion and trafficking. The RAB family of small G proteins regulates intercellular vesicle trafficking, including exocytosis, endocytosis, and recycling	Unknown
	Sult1e1	sulfotransferase family 1E, member 1	Catalyze the sulfate conjugation of many hormones, neurotransmitters, drugs, and xenobiotic compounds	Unknown
mmu-miR-149-3p	Apoc2	Apolipoprotein C-II	Extracellular region or secreted. Hydrolyzes triglycerides and thus provides free fatty acids for cells	o
	Rab11fip5	Rab11 family interacting protein 5	Rab effector involved in protein trafficking from apical recycling endosomes to the apical plasma membrane	Unknown
	Leng8	Leukocyte receptor cluster member 8	Predicted using known transcription factor binding site motifs	Unknown
	Galnt17	Polypeptide N-acetylgalactosaminyl transferase 17	Serve a variety of structural and functional roles in membrane and secreted proteins	Unknown

Improvement of stem cell-derived exosome release efficiency by surface modified nanoparticles

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