PLGA-PEI and PLGA-Cy5.5 materials
PLGA (50:50, MW 38,000-54,000; Sigma-Aldrich, St. Louis, MO, USA) and PEI (MW ~ 25,000, Mn ~ 10,000; Sigma-Aldrich) were used to synthesize the PLGA-PEI copolymer. N,N’-Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were prepared as linkers between PLGA and PEI. PLGA (330 mg), PEI (113.67 mg), DCC (18.33 mg), and NHS (11 mg) 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 .
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 dropwise 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 negatively charged, surface-modified NPs. EDC and NHS were prepared as a linker between modified NPs and 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 by purification, therefore, are not expected to cause any side effects in this study. 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 produce negatively charged NPs. In addition, positively charged NPs have also been made using the 8:2 ratio of PLGA:PEI.
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 dropwise to 3 mL of deionized water. Next, 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  and −14 mV for FluidMAG-CMX .
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 evaluate the internal SPIO core, TEM measurements were performed. The samples for TEM were prepared by the drop casting method over a carbon grid as previously described [15, 25].
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 alpha-MEM 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
GFP-labeled MSCs were plated in 60 mm culture dishes at 1×105 cells/well. After 24 h incubation period with PCS NPs, the cells were washed twice with PBS. Next, samples were analyzed by flow cytometry using a FACS Aria3 flow cytometer (Becton Dickinson, San Jose, CA, USA). The data were analyzed using the FACS Diva software.
Cell viability was measured by counting viable cells using the EZ-3000 CCK kit (Dogen, Seoul, Korea) (CCK-8) according to the manufacturer’s instructions. Briefly, the MSCs were seeded into 96-well plates (5×102 cells/well) 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 for 24h, 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 of incubation.
MSCs and GFP-labeled MSCs were seeded (5×103 cells/well) into 4-well cell culture plates. After 24 h of incubation, the culture medium was replaced with fresh medium and NPs were added. After incubation for 24 h with NPs, the MSCs were washed twice with PBS, 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 also stained with specific antibodies. The following primary antibodies were used: 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 also used to confirm the localization 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.
Two inhibitors of NP internalization were used: Dynasore (ab120192, Abcam) and Pitstop2 (SML-1169, Sigma-Aldrich). Both inhibitors were used at a concentration of 40 μM/dish and they remained active 3 h prior to NP treatment. Dynasore inhibits dynamin, an important factor in receptor-mediated endocytosis, while Pitstop2 blocks 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 used to block Rab7 by competitive inhibition.
Cells were fixed in 2.5% glutaraldehyde for 2 h at 4 °C 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 stepwise manner using 50% to 100% ethanol gradient. Next, the samples were embedded in Epon resin. Ultrathin sections were cut using an ultra-microtome and stained with uranyl acetate and lead citrate. TEM was performed at 63 kV.
Preparation of exosomes
Exosomes from the medium of cultured MSCs were isolated using Total Exosome Isolation reagent (4478359; Invitrogen, Carlsbad, CA, USA), strictly according to the manufacturer’s instructions. PBS was used to resuspend the purified exosomes. Exosomes were stored 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 X-100 in PBS and a protease inhibitor cocktail.
Real-time reverse transcription polymerase chain reaction (qRT-PCR)
To prepare mRNA samples, a total volume of 10 µL, including 2 µL of mRNA and 8 µL of reverse transcriptase mix, 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 for the cDNA synthesis kit (TOYOBO, Osaka, Japan), samples were incubated at 42°C for 60 min and deactivated at 95°C for 5 min. Next, cDNA was diluted 1:10 with 90 µL nuclease-free water for miRNA qRT-PCR.
qRT-PCR was performed using Applied Biosystems 7900 thermal cycler (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. qPCR cycle conditions and steps were as follows: pre-amplification at 95°C for 3 min, 40 cycles of 95°C for 10 s, 60°C for 60 s, and melting curve analysis. All qPCR assays were performed in duplicate. The mRNA expression levels were normalized 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 spectrophotometer 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 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 using species-specific annotated probes. The comparative analysis between the test and control samples was carried out using fold change. All statistical testing and visualization of differentially expressed genes were conducted using R statistical language 3.3.3 (https://www.r-project.org/).
MSCs were sub-cultured, plated in a 60 mm dish at 2×10 5 cells/dish, and incubated for 24 h. The next day, the NPs were added and a magnet of the same size as the plate was immediately placed under the bottom of the plate. The cells were incubated for 1, 6, or 24 h, and then washed with PBS. Cells were detached using trypsin and the mean intensity value was measured using FACS in a round vial. For TEM, electron microscopy images were analyzed as described below.
Imaging analysis quantification of exosome amounts
To quantify the number of exosomes using image analysis, the images of cells expressing exosomes with a size of 50 to 150 nm were obtained. Equations (1) and (2) were created to quantify the number of exosomes. The value definition within the equation is as follows:
qPCR results were analyzed to compare the mRNA levels of autophagy markers. The 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 using GraphPad PRISM version 5.0 (GraphPad Inc., La Jolla, CA, USA). The sensitivity and specificity of the statistically significant mRNAs were analyzed using GraphPad PRISM version 5.0. P-values < 0.05 were considered statistically significant.