Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Magnetically Navigated Intracellular Delivery of Extracellular Vesicles Using Amphiphilic Nanogels Ryosuke Mizuta,† Yoshihiro Sasaki,*,† Riku Kawasaki,†,‡ Kiyofumi Katagiri,§ Shin-ichi Sawada,† Sada-atsu Mukai,† and Kazunari Akiyoshi*,†
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Department of Polymer Chemistry, Graduate School of Engineering, A3-317, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan ‡ RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan § Department of Applied Chemistry, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan ABSTRACT: Various cells in vivo secrete exosomes consisting of lipid bilayers. They carry mRNAs and miRNAs capable of controlling cellular functions and can be used as drug delivery system nanocarriers. There is the current need to further improve the efficiency of exosome uptake into target cells. In this study, we prepared a hybrid of exosomes and magnetic nanoparticles, which could be guided to target cells by a magnetic field for efficient uptake. Magnetic nanogels were prepared and hybridized to fluorescently labeled exosomes isolated from PC12 cells. By applying a magnetic field to a hybrid with magnetic nanogel, exosomes were efficiently transferred into target cells as confirmed by confocal laser microscopy. Finally, we found that differentiation of adipose-derived stem cells to neuron-like cells was enhanced by magnetic induction of the exosome-magnetic nanogel hybrid, indicating maintenance of the intrinsic functions of the exosomes in the differentiation of adipose-derived stem cells.
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peptide, which improves the efficiency of delivery into cells.10 In this manner, approaches to improve the intracellular delivery efficiency of exosomes have been reported, but the number is still small. Here, we report a method to efficiently deliver exosomes to target cells using a magnetic field. This is the first example demonstrating delivery of exosomes using an external magnetic field. For this purpose, we prepared a hybrid of magnetic nanoparticles and exosomes. In recent years, the magnetic drug delivery system has attracted attention. This system has excellent target specificity and can reduce the amount of drug required to achieve a particular concentration in the vicinity of the target.11,12 Magnetic force is noninvasive, and its strength and direction are easy to control. An example of combining exosomes and magnetic nanoparticles has been reported previously. Indeed, Qi et al. reported modifying exosomes with transferrin-conjugated magnetic nanoparticles for efficient isolation of exosomes from blood.13 They targeted the transferrin receptor present in the exosome membrane, efficiently dissociated exosomes by magnetizing, and enabled targeting to tumors in vivo. In addition, Silva and colleagues loaded iron oxide nanoparticles inside exosomes using the phagocytosis of macrophages and produced magnetoresponsive exosomes.14 This complex of magnetic nanoparticles and exosomes allowed excellent magnetic targeting and magnetic
INTRODUCTION Exosomes are secreted from various cells constituting the body as a transmission medium for intercellular communication.1 Because the contents of exosomes are specific to the releasing cells, they can be biomarkers of diseases.2 Recently, it has been reported that exosomes contain substances such as mRNAs and miRNAs capable of controlling cellular functions. Thus, exosomes are attracting attention in advanced medical fields including regenerative medicine.3 miRNAs play important roles in various cellular functions such as cell proliferation and differentiation. Let-7a belonging to the let-7 family exerts an inhibitory effect on tumor growth in breast cancer.4 Furthermore, exosomal miRNAs induce the differentiation of stem cells.5 Because exosomes consist of cell membrane-derived lipid bilayer membranes, which have excellent biocompatibility, Studies are underway to apply exosomes as drug delivery system (DDS) nanocarriers. For example, there are reported methods to encapsulate siRNA and anticancer drugs,6,7 and to transfer functional membrane proteins directly into cellular membranes.8 When applying exosomes as a DDS carrier, development of a technique to further improve the efficiency of exosome uptake into target cells is desired. In a recent study, it was reported that exosomes can be delivered to cells efficiently by binding cationic lipids and GALA peptides to exosomes.9 Cationic lipids promote intracellular delivery by relieving the charge repulsion of exosomes and the target cell. Furthermore, macropinocytosis is induced by modifying the exosome surface with an arginine-rich membrane-permeable © XXXX American Chemical Society
Received: May 24, 2019 Revised: July 16, 2019 Published: July 19, 2019 A
DOI: 10.1021/acs.bioconjchem.9b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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induction using this hybrid. In addition, to confirm whether efficient intracellular delivery was carried out while maintaining the functions of exosomes, functional evaluation of the hybrid was carried out using an exosome derived from PC12 cells. This PC12 exosome is reported to induce differentiation of mesenchymal stem cells into neural-like cells by internal miRNA.20
resonance imaging in vivo. Although reports have focused on magnetic targeting of complexes of magnetic nanoparticles and exosomes, few reports have improved the efficiency of delivery to target cells by magnetic induction. In addition, when complexing magnetic nanoparticles with exosomes, the original function of exosomes should be maintained for application to therapy. Therefore, in this study, we investigated a method to bind exosomes and magnetic nanoparticles via noncovalent and hydrophobic interactions. When preparing a hybrid of exosomes and magnetic nanoparticles, we used a magnetic nanogel that we reported previously.15 A magnetic nanogel is a complex of an amphiphilic nanogel consisting of cholesterolbearing pullulan (CHP) and iron oxide nanoparticles. We have reported that magnetic nanogels have excellent magnetic induction capability and that biomolecules can be delivered into cells with extremely high efficiency using magnets. In addition, this magnetic nanogel did not show apparent cytotoxicity.16 We also reported that a CHP nanogel forms a stable complex with an artificial lipid bilayer and plasma membrane via its cholesteryl group.17−19 When hydrophobic cholesteryl groups in nanogels approach the lipid membrane, the CHP nanogel forms a stable complex via hydrophobic interactions by inserting its cholesteryl groups into the lipid membrane. Based on this background, we designed a hybrid consisting of exosomes and magnetic nanogels (Figure 1).
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RESULTS AND DISCUSSION Hybridization of Exosomes and Magnetic Nanogels. Exosomes were isolated from the culture medium of PC12 cells by the most common ultracentrifugation method. After resuspending the isolated exosomes in PBS, the particle size distribution was measured by nanoparticle tracking analysis (NTA) (Figure 2A). Based on the results of NTA, the average
Figure 2. Characterization of PC12 cell-derived exosomes. (A) Size distribution profile of exosomes determined by NTA. (B) Morphological observation by TEM. Scale bar represents 200 nm.
particle size was about 150 nm. Morphological observation was also performed by transmission electron microscopy (TEM). As a result, it was possible to observe exosomes of about 150 nm, which supported the NTA results. Moreover, it was confirmed that the exosomes were hollow spheres surrounded by a lipid membrane (Figure 2B). To deliver exosomes more efficiently to target cells while maintaining their functions, we designed a magnetic delivery system in which we prepared a hybrid consisting of exosomes and a magnetic nanogel. In this system, the magnetic nanogel and exosomes were first compounded, and then a magnetic field was applied using a neodymium magnet. Magnetic nanogels were prepared according to previously reported methods.15 Specifically, CHP nanogels were formed using cholesteryl-group-substituted pullulan (100 μg/mL). The average particle diameter of a CHP nanogel was 34 nm. Thereafter, magnetic nanogels were prepared by mixing oleicacid-coated iron oxide (100 μg/mL) with a CHP nanogel. The average particle size of the prepared magnetic nanogel was measured by dynamic light scattering (DLS) and found to be about 120 nm. Compared with the particle diameter of the CHP nanogel, the particle size of the magnetic nanogel was larger, but this result appeared to be caused by iron oxide nanoparticles gathering at the center of the nanogel. Hybrids were prepared by mixing PKH67-stained exosomes derived from PC12 cells and magnetic nanogels, followed by incubation at 37 °C. To confirm hybridization, the hybrid was separated using a magnetic field at each time after mixing
Figure 1. Schematic diagram of intracellular exosome delivery by magnetic induction.
Because the hybrid of the magnetic nanogel and exosomes has a magnetoresponsive ability that does not modify the inside of exosomes or its membrane proteins, it was expected that the exosomes could be efficiently delivered to target cells by magnetic induction without affecting the original function of the exosome. In this study, we investigated efficient delivery of exosomes to human adipose derived mesenchymal stem cells by magnetic B
DOI: 10.1021/acs.bioconjchem.9b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry exosomes and magnetic nanogels, and the fluorescence intensity of free exosomes was quantified in the supernatant. The ratio of hybridization was calculated based on the initial fluorescence intensity of exosomes, which revealed that about 80% complexed within 24 h (Figure 3A). In addition, the final
laser microscope. Fluorescence of the magnetic nanogels and exosomes was confirmed (Figure 4B). The results showed that the system in which the magnetic field was applied to the hybrid had stronger fluorescence of exosomes and magnetic nanogels than exosome only. Therefore, by applying a magnetic field to the hybrid, the exosomes were efficiently transferred into the target cells. In addition, images acquired by confocal laser scanning fluorescence microscopy (CLSFM) suggested that exosomes derived from PC12 cells were effectively internalized in almost all cells via the magnetic nanogels. Intracellular uptake efficiencies were compared by applying magnetic fields in different orientations (Figure 4C). The magnet was placed on the top, side, or bottom of the dish. Exosomes were taken up most when the magnet was placed on the bottom of the dish. The exosomes complexed with the magnetic nanogel, suggesting that the magnetic nanogel responded to the magnetic field and was attracted to the magnet together with the exosomes. Thus, when the magnet was placed under the dish, exosomes were efficiently delivered to the target cells. Under the same conditions, flow cytometry showed that the amount of exosome uptake at 24 h after induction by a magnetic field was increased markedly compared with exosomes only or without the magnetic field applied to the hybrid (Figure 4D). After adding the hybrid and applying a magnetic field for 24 h, cell viability was compared with conditions without the magnetic field or exosomes only (Figure 4E). No decrease in the survival rate was observed when a magnetic field was applied to the hybrid, suggesting that the cytotoxicity of the exosome−magnetic nanogel hybrid was low. Therefore, this hybrid was highly biocompatible and represented an excellent exosome delivery system. Control of Cellular Functions by Magnetically Delivered Exosomes. The ability of the magnetic delivery system to control cellular functions was evaluated by induction of neuron-like differentiation in adipose-derived mesenchymal stem cells. Recently, mesenchymal stem cells have been applied to regenerative medicine. In addition, exosomes are reported to be one of the factors that cause mesenchymal stem cells to differentiate into various cell types. Therefore, it is essential to develop a method to deliver exosomes more efficiently while maintaining their function. Neuron-like differentiation is induced by miR-125b that is contained in exosomes isolated from differentiating rat pheochromocytoma (PC12) cells.20 After delivering exosomes into adipose derived mesenchymal stem cells (ADSCs) by magnetic induction and culture for 1 week, differentiation of ADSCs to neuron-like cells was confirmed by immunostaining. We used MAP2 as a differentiation marker of mature neurons. As shown in Figure 5, the fluorescence appeared to be stronger when exosomes were delivered by applying a magnetic field to the hybrid of PC12 cell-derived exosomes and the magnetic nanogel compared with conditions without the magnetic field or exosomes only. Therefore, differentiation was progressing compared with the system in which only exosomes were added, because exosomes were efficiently delivered into the target cells by the exosomemagnetic nanogel hybrid. Moreover, neurite outgrowth was prominent in the system in which the magnetic field was applied to the hybrid, and exosomes delivered by magnetic induction further differentiated neuron-like cells. Therefore, improvement of the delivery efficiency was achieved while maintaining the intrinsic function of the exosomes by the
Figure 3. Hybridization of exosomes and magnetic nanogels. (A) Time dependence of hybridization. (B) Magnetic nanogel concentration dependence of hybrids (exosomes with protein concentration of 50 μg/mL at various weight ratios of magnetic nanogels).
concentration of exosomes was fixed at 50 μg/mL, the concentration of the magnetic nanogel was changed, and by confirming the hybridization rate after 24 h, the dependence of the magnetic nanogel concentration was confirmed (Figure 3B). As a result, exosomes with a protein concentration of 50 μg/mL were sufficiently hybridized with about 100 μg/mL magnetic nanogel. The average particle size of the hybrid of magnetic nanogel and exosome was about 230 nm, and the particle size of the hybrid after 24 h incubation was about 250 nm, indicating the hybrid would be colloidally stable. Magnetic Induction Delivery of Exosomes. Exosome magnetic nanogel hybrids were prepared using exosomes and rhodamine-modified magnetic nanogels. The hybrids were magnetically induced to target cells to confirm whether exosomes could be delivered efficiently. The hybrid was added to medium, and a magnet was placed on the bottom of the dish for 24 h, followed by observation using a confocal C
DOI: 10.1021/acs.bioconjchem.9b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 4. Cellular uptake of exosome−magnetic nanogel hybrids by magnetic induction. (A) Schematic representing the direction of the applied magnetic field. (B) CLSFM images of human mesenchymal stem cells (ADSCs). Hybrid (0.5 T) represents cells in which the exosome−magnetic nanogel hybrid was added and a magnetic field was applied by a magnet having a magnetic flux density of 0.5 T. Hybrid (0 T) was a cell in which the exosome−magnetic nanogel hybrid was added and incubated without applying a magnetic field. The magnet was placed on the bottom of dish for 24 h. Scale bar represents 100 μm. (C) CLSFM images of ADSCs incubated with exosome-magnetic nanogel hybrid subjected to magnetic fields in different orientations. (D) Cellular uptake of PKH67 labeled exosome determined by flow cytometry at 24 h. (E) Cell viability of exosome−magnetic nanogel hybrid.
magnetic induction maintained their functions and induced the differentiation of adipose-derived mesenchymal stem cells into neuron-like cells. This approach may be an excellent method of delivering exosomes to a target site in the body as a therapy. In addition, the amount of exosomes used was smaller than that in the previously reported method,20 indicating that this approach may be effective to analyze the function of a trace amount of exosomes.
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METHODS
Cell Culture. The rat pheochromocytoma cell line PC12HS (JCRB0266; JCRB Cell Bank, Japan), which is a clone highly sensitive to nerve growth factor (NGF), was cultured in DMEM supplemented with 2% heat-inactivated horse serum (Thermo Fisher Scientific) and penicillin−streptomycin on poly-L-lysine-coated dishes. Human adipose-derived mesenchymal stem cells (hADSCs; Lonza) were cultured in ADSC-1 (KOHJIN BIO) using a poly-L-lysine-coated glass-bottomed dish. All cells were cultured in a humidified incubator at 37 °C with 5% CO2. Exosome Isolation and Characterization. Exosomes were isolated by ultracentrifugation. Briefly, PC12 was incubated in exosome-free FBS that had been centrifuged at 100 000g before use. The culture supernatant was collected, centrifuged at 300g for 10 min to remove cells, at 2000g for 20 min, and finally at 10 000g for 45 min (all at 4 °C), followed by filtration through a 0.22 μm filter. Exosomes were pelleted by ultracentrifugation at 120 000g for 70 min at 4 °C. The pellets
Figure 5. Neuron-like differentiation of human adipose derived mesenchymal stem cells (hADSCs) by magnetically guided exosomes. Immunofluorescence staining of the neuronal differentiation marker protein microtubule-associated protein 2 (MAP2). Scale bar indicates 200 μm.
noncovalent formation of the hybrid of exosomes and magnetic nanogel.
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CONCLUSION A magnetic nanogel formed a stable hybrid with exosomes. By applying a magnetic field to this hybrid, exosomes were delivered to the target cells with extremely high efficiency. Furthermore, PC12 cell-derived exosomes delivered by D
DOI: 10.1021/acs.bioconjchem.9b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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respectively. ADSCs were plated in a glass-bottomed dish (Iwaki GLASS, Japan) at a density of 3 × 103 cells/well and cultured at 37 °C with 5% CO2 overnight. PKH67-labeled exosomes (protein concentration: 10 μg/mL) or exosome− magnetic nanogel hybrids (protein concentration: 10 μg/mL) were added to ADSCs, and the cells were cultured for 24 h. Hybrid (0.5 T) represents cells in which the exosome− magnetic nanogel hybrid was added and a magnetic field was applied by a magnet having a magnetic flux density of 0.5 T. Hybrid (0 T) was a cell in which the exosome−magnetic nanogel hybrid was added and incubated without applying a magnetic field. The viability of each group was measured using a cell counter after staining with trypan blue. The cells were fixed with 4% paraformaldehyde for 15 min and then washed with PBS. Cells were then observed by CLSFM. ADSCs were seeded on a glass-bottomed dish at a density of 3 × 103 cells/ well, and the hybrid was added to cells under the influence of a magnetic field. The magnet was placed on the top, side, or bottom of the dish. A total of 1 × 105 ADSCs incubated with PKH67 labeled exosomes or exosome−magnetic nanogel hybrids for 24 h at 37 °C were washed twice with PBS, detached using trypsin, and resuspended in BD buffer. Flow cytometry was performed on a Cytomics FC500 flow cytometer (Beckman Coulter). Immunofluorescence Staining. ADSCs were seeded on a poly-L-lysine-coated glass-bottomed dish at a concentration of 1.5 × 103 cells/dish and cultured overnight. PC12 cells were maintained in DMEM containing 1% penicillin−streptomycin, 2% horse serum, and 25 ng/mL NGF to induce neuronal differentiation, and then exosomes were isolated from the culture supernatant. ADSCs were exposed to exosomes derived from PC12 cells (protein concentration: 10 μg/mL) or magnetic nanogel−PC12−exosome hybrids (protein concentration of exosomes: 10 μg/mL; concentration of magnetic nanogel: 100 μg/mL). After 24 h of magnetic induction, the medium was changed to fresh DMEM, which was repeated every day. At 7 days, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized in PBS containing 0.5% Triton-X for 7 min. After blocking with PBS containing 1% bovine serum albumin for 1 h, anti-NSE and anti-MAP2 antibodies were applied to the cells at 4 °C overnight. After washing with PBS, a secondary antibody conjugated with Alexa Fluor 488 was applied for 1 h. Nuclei were counterstained with Hoechst 33342. The samples were observed by CLSFM (LSM780).
were rinsed with phosphate-buffered saline (PBS), ultracentrifuged again, and dispersed in PBS. The protein concentrations of the exosome suspensions were determined using a BCA (bicinchoninic acid) assay (Thermo Scientific, Waltham, MA). The size distribution of the exosomes was analyzed using a Nano Sight LM10 instrument (Marvern instruments Ltd., Worcestershire, UK) with NTA software version 2.3. Proteins (6 μg) of exosomes fixed with 4% paraformaldehyde were applied to Formvar-coated grids (PVFC10) for 15 min. Samples were stained with 1% phosphotungstic acid and observed under a HT7700 transmission electron microscope (Hitachi, Japan) at an accelerating voltage of 100 kV. Preparation of the Exosome−Magnetic Nanogel Hybrid. The magnetic nanogel was prepared as described previously.15 Briefly, CHP was suspended in Milli-Q water and stirred overnight at room temperature. The solution was sonicated using a probe-type sonicator (Sonifier 250, Branson, Danbury, CT) at 40 W for 15 min with cooling on ice. The solution was filtered through a 0.22 μm polyvinylidene difluoride filter (Millex-HV, Millipore, Bedford, MA). Oleicacid-coated Fe3O4 nanoparticles were synthesized via a conventional hydrothermal process and purified by removing byproducts in n-hexane with centrifugation. The Fe3O4 nanoparticles were then redispersed in tetrahydrofuran (THF; 2 mg/mL−1). The magnetic nanogel was obtained by injecting the THF suspension of the oleic-acid-coated Fe3O4 nanoparticles into a CHP nanogel aqueous suspension with vortex mixing. The solution was lyophilized to remove THF and redispersed in PBS. The basic physicochemical properties for the magnetic nanoparticles have already reported elsewhere.15 Hybridization of exosomes and the magnetic nanogel was confirmed after magnetic separation of the hybrid from free exosomes. PKH67 (Sigma, USA) dye was diluted in diluent C to a final concentration of 4 μM (dye solution). The dye solution was added to the exosome suspension and allowed to stand at room temperature for 5 min. Next, it was ultracentrifuged at 120 000g for 70 min at 4 °C and resuspended in PBS. The magnetic nanogel and PKH67stained exosomes were mixed and incubated for 24 h at 37 °C. The final concentrations of the magnetic nanogel and exosomes were 100 and 50 μg/mL, respectively. At each time point (0, 0.5, 1, 8, 24 h after mixing), exosomes incorporated into the magnetic nanogel were magnetically separated from free exosomes, and the complexation ratio was determined according to the following equation: Ratio of hybridization (%) = (1 − f/f 0) × 100, where f and f 0 are the fluorescence intensity from released exosomes and whole exosomes, respectively. The hybridization ratio of magnetic nanogel and exosome was assessed by measuring the fluorescence intensity in a fluorescence spectrometer (FP6500, JASCO, Japan, Tokyo). An exosome suspension (50 μg/ mL as the final concentration) in PBS was mixed with the magnetic nanogel at various concentrations and incubated for 24 h at 37 °C. Then, exosomes incorporated into the magnetic nanogel were magnetically separated from free exosomes, and the hybridization ratio was determined. Delivery of Exosome−Magnetic Nanogel Hybrids. Intracellular localization of magnetic nanogel−PKH67-labeled exosome hybrids was observed by CLSFM (LSM780). The magnetic nanogel and PKH67-stained exosomes were mixed and incubated for 24 h at 37 °C. The final concentrations of the magnetic nanogel and exosomes were 100 and 10 μg/mL,
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +81-75383-2823. Fax: +81-75-383-2590. ORCID
Yoshihiro Sasaki: 0000-0003-1333-5347 Kiyofumi Katagiri: 0000-0002-9548-9835 Sada-atsu Mukai: 0000-0001-9909-9116 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Grant-in Aid from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP16H06313 (K.A.) and JP16H03842 (Y.S.), and by Training Program of Leaders for Integrated Medical System for E
DOI: 10.1021/acs.bioconjchem.9b00369 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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(14) Silva, A. K. A., Kolosnjaj-Tabi, J., Bonneau, S., Marangon, I., Boggetto, N., Aubertin, K., Clement, O., Bureau, M. F., Luciani, N., Gazeau, F., et al. (2013) Magnetic and photoresponsive theranosomes: Translating cell-released vesicles into smart nanovectors for cancer therapy. ACS Nano 7 (6), 4954−4966. (15) Katagiri, K., Ohta, K., Sako, K., Inumaru, K., Hayashi, K., Sasaki, Y., and Akiyoshi, K. (2014) Development and potential theranostic applications of a self-assembled hybrid of magnetic nanoparticle clusters with polysaccharide nanogels. ChemPlusChem 79 (11), 1631−1637. (16) Kawasaki, R., Sasaki, Y., Katagiri, K., Mukai, S., Sawada, S., and Akiyoshi, K. (2016) Magnetically guided protein transduction by hybrid nanogel chaperones with iron oxide nanoparticles. Angew. Chem., Int. Ed. 55 (38), 11377−11381. (17) Ueda, T., Lee, S. J., Nakatani, Y., Ourisson, G., and Sunamoto, J. (1998) Coating of POPC giant liposomes with hydrophobized polysaccharide. Chem. Lett. 27, 417−418. (18) Sekine, Y., Moritani, Y., Ikeda-Fukazawa, T., Sasaki, Y., and Akiyoshi, K. (2012) A hybrid hydrogel biomaterial by nanogel engineering: bottom-up design with nanogel and liposome building blocks to develop a multidrug delivery System. Adv. Healthcare Mater. 1 (6), 722−728. (19) Bal, T., Oran, D. C., Sasaki, Y., Akiyoshi, K., and Kizilel, S. (2018) Sequential Coating of Insulin Secreting Beta Cells within Multilayers of Polysaccharide Nanogels. Macromol. Biosci. 18 (8), 1800001. (20) Takeda, Y. S., and Xu, Q. B. (2015) Neuronal differentiation of human mesenchymal stem cells using exosomes derived from differentiating neuronal cells. PLoS One 10 (8), No. e0135111.
Fruitful Healthy-Longevity Society (LIMS), Program for Leading Graduate Schools, MEXT, Japan. This work was also supported by a grant from JST CREST Grant Number JPMJCR17H2. We thank Mitchell Arico from the Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
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ABBREVATIONS
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REFERENCES
miRNA, micro RNA; mRNA, mRNA; siRNA, small interfering RNA; DDS, drug delivery system; CHP, cholesterol groupbearing pullulan; NTA, nanoparticle tracking analysis; TEM, transmission electron microscopy; CLSFM, confocal laser scanning fluorescence microscopy; NGF, nerve growth factor; PBS, phosphate-buffered saline; ADSC, apidose derived stem cells; DMEM, Dulbecco’s modified Eagle’s medium
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