Cell tradition
The human OS cell strains 143B, MG63, Saos-2, and U2-OS (Procell, China) have been subjected to tradition in a high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone, USA) that contained 10% fetal bovine serum (FBS) (HyClone, USA) and 1% penicillin/streptomycin (HyClone, USA) at 37 °C in 5% CO2. Human BMSCs (Hm-BMSCs, Procell, China) went by way of tradition in alpha-MEM (Hyclone, USA) containing 10% FBS (HyClone, USA) and 1% penicillin/streptomycin (HyClone, USA) at 37 °C in 5% CO2.
EXO preparation
The purification of EXO was carried out following beforehand established protocols, albeit with sure adjustments [47]. The conditioned cell tradition medium that contained EXO was subjected to fifteen min centrifugation at 3,000 g at 4 °C, aiming at eliminating cells. The ensuing supernatant was subjected to 60 min centrifugation at 100,000 g at 4 °C, washing with chilly PBS, and one other 60 min centrifugation at 100,000 g at 4 °C. EXO was resuspended in PBS and preserved at -80 °C.
The research decided EXO focus by measuring protein content material by way of Pierce BCA Protein Assay Equipment (Thermo, USA). The EXO samples have been diluted to a focus of 1 mg/mL and subjected to dimension distribution evaluation using the Mastersizer instrument (Malvern, UK). The research employed transmission electron microscopy (TEM) (Hitachi HT-7800, Japan) to look at the morphology. Moreover, Western blotting (WB) was utilized for figuring out EXO marker expressions, CD9/63/81, and TSG101.
Modification of EXO with bone-targeting peptide
The Sener Organic Expertise (Hefei, China) synthesized bone-targeting-peptide (98% purity assayed by HPLC), which was then dissolved in an answer of 5 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) at 1 mg/mL. Subsequently, Xi’an Rixi Organic Expertise Firm (Xi’an, China) synthesized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG-Mal, 98% purity assayed by HPLC), which was then dissolved in a 5 mM HEPES buffer at 1 mg/mL. DSPE-PEG-Mal was subsequently utilized to react with the peptide in a molar ratio of 1:1 at room temperature for twenty-four h, aiming at making ready DSPE-PEG-Mal-Cys-SDSSD. The combination went by way of 24 h dialysis in opposition to distilled water utilizing a dialysis bag (molecular weight cutoff of 3000 Da). Finally, the resultant post-dialysis answer skilled freeze-drying, following which the DSPE-PEG-Mal-Cys-SDSSD powder was preserved at − 20℃ [7].
For the aim of mixing EXO with bone-targeting peptide, an answer consisting of 10 μL of EXO (1012 particles/mL) and 90 μL of the DSPE-PEG-Mal-Cys-SDSSD (10 μM) was gently launched into 100 μL PBS. The ensuing combination was then subjected to an in a single day incubation at 4℃. For the removing of uncombined DSPE-PEG-Mal-Cys-SDSSD, the combination was subjected to PBS washing by 70 min ultracentrifugation at 105 ×g at 4℃ and was then resuspended in PBS [7].
Drug loading into BT-EXOs
The drug loading course of occurred following the beforehand described methodology [48]. Briefly, the mixture of BT-EXO and CAP was subjected to sonication utilizing specified settings because the experimental protocol concerned the applying of 20% amplitude for a complete of 6 cycles consisting of 30 s on/off for 3 minutes with a two-minute cooling interval between every cycle. The ensuing BT-EXO-CAP answer was subjected to 60 min incubation at 37 °C to allow the exosomal membrane restoration in addition to to remove any excess-free drug by way of ultracentrifugation. The research employed UV-Spectroscopy (Omega POLARstar, Germany) to measure the free CAP focus within the supernatant on the RF λmax (268 nm) [49].
Drug launch assay
The experiment concerned immersing dialysis tubes in PBS options with pH values of seven.4 and 4.5, adopted by the dedication of the in vitro drug launch profile by transferring 3 mL of BT-EXO-CAP answer into the tubes [50].The discharge of CAP into the showering medium was assessed at 37 °C over 48 h on the RF λmax (268 nm) utilizing UV-Spectroscopy (Omega POLARstar, Germany) at time intervals of 1, 2, 4, 6, 12, 18, 24, 36, and 48 h.
Circulate cytometry
The cell tradition was maintained in six-well plates and subjected to numerous remedies for twenty-four h. Following this, the cells have been collected and subjected to 30 min staining with FerroOrange dye (#F374, Dojindo Laboratories) or 10 μM C11-BODIPY™ 581/591 (#D3861, Thermo Fisher Scientific, USA). The cells have been uncovered to in a single day fixation in chilly 70% ethanol at − 20 °C, adopted by staining with propidium iodide for the aim of conducting cell cycle evaluation. Circulate cytometry was performed (Beckman Cytoflex LX, USA), and information have been analyzed by CytExpert software program.
Cytotoxicity assay
The experiment concerned seeding cells into 96-well plates (1 × 104 cells/nicely), adopted by therapy as specified. The absorbance of lowered WST-8 at 450 nm was measured at totally different time intervals for calculating the cell numbers in three replicate wells.
Colony formation
The measurement of colony formation was performed following the beforehand outlined methodology [51]. Six-well plates have been utilized for cell seeding (200 cells/nicely), and the cells have been then subjected to 7-day tradition and 20 min fixation with ice-cold methanol previous to being stained with crystal violet. Colonies containing > 50 cells have been counted using an optical microscope (Carl Zeiss, Germany).
Wound therapeutic assay
Briefly, 5 × 105 cells went by way of 24 h tradition in six-well plates, and a 200 μL sterile pipette was used for scratching the monolayers, adopted by taking the pictures after 24 h. The experiment was repeated 3 times.
Transwell assay
Matrigel answer was utilized to the higher chamber for two h (Corning, Australia) whereas including a 1 × 105 cells/nicely cultured in 100 μL FBS-free medium with 600 μL full medium into the decrease chamber. After 24 h, non-migrating cells located on the higher facet of the chamber have been eradicated. The invasive cells situated within the decrease chamber have been subjected to twenty min fixation with methanol and subsequent 30 min staining with 0.1% crystal violet (Solarbio, China) previous to being counted by way of an inverted microscope.
RNA sequencing
The RNA extraction course of concerned the usage of TRIzol to isolate whole RNA from three distinct OS cells. Subsequently, RNA-Seq was performed on the extracted RNA, and the ensuing information have been analyzed on the UAB Genomics Core Facility. The RNA-Seq was performed utilizing the Illumina NextSeq500 platform, following the protocols (Illumina Inc., San Diego, CA).
Intracellular ROS measurements
The intracellular ROS measurement was performed by way of DCFDA/H2DCFDA-Mobile ROS Assay Equipment (Abcam, USA). Briefly, cells have been subjected to 30 min incubation with 20 μM DCFDA at 37 °C. Following this, the cells have been washed with PBS, and the ensuing fluorescence was detected utilizing an Olympus BX50 fluorescence microscope.
WB evaluation
The experiment concerned the separation of equal portions of protein per nicely by way of the usage of 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated protein was then transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes went by way of incubation with main antibodies that have been particularly raised in opposition to the next proteins: β-Actin (1:3000, Cat#ab20272, Abcam), CD63 (1:1000, Cat#ab68418, Abcam), TSG101 (1:1000, Cat#ab125011, Abcam), CD9 (1:1000, Cat#ab236630, Abcam), CD81 (1:1000, Cat#ab79559, Abcam), calnexin (1:1000, Cat#ab22595, Abcam), ACSL4 (1:10000, Cat#ab155282, Abcam), TFRC(1:1000, Cat#ab214039, Abcam), ALOX12(1:1000, Cat#ab168384, Abcam), GPX4(1:1000, Cat#ab125066, Abcam), SLC7A11(1:1000, Cat#ab307601, Abcam), FTH1(1:1000, Cat#ab75972, Abcam), Keap1(1:1000, Cat#ab227828, Abcam), Nrf2(1:1000, Cat#ab62352, Abcam), PCNA(1:5000, Cat#ab92552, Abcam).
Mobile GSH ranges
The GSH measurement was performed by a equipment, per the protocols (Beyotime, China). The cells have been cultured in 6-well plates, adopted by including lysate and centrifuging the medium at 10,000 g for 10 min. The GSH focus within the supernatant was decided by referencing a typical curve.
Nrf2 overexpression lentivirus transfection
The 143B cells within the logarithmic progress stage went by way of seeding into 6-well plates. Upon reaching 60% cell density, 2 μL GFP-NC or GFP-Nrf2 lentivirus answer (Haixing Biosciences) was launched into the cell medium, respectively. After 24 h, the Nrf2 stably overexpressed cells have been screened by the addition of puromycin at a focus of two μg/ml.
Animal experiments
The research utilized male BALB/c nude mice (5-week-old) procured from Xiamen College Laboratory Animal Heart (Xiamen, China). The mouse have been housed in a temperature-controlled atmosphere (24 ± 1℃) with a 12 h mild/12 h darkish cycle, and an air filter cowl was offered. The under-anesthesia mice obtained an injection of 143B cells (2 × 106 /10 μL) into the proper tibia and have been allowed to develop for 7 days, aiming at setting up the nude mouse OS mannequin, whereas injecting saline with BT-EXO (1 × 108/mL) and CAP (20 mg/kg) by way of the tail vein. A complete of 68 mice have been divided into 4 teams for the experiment. The Xiamen College Laboratory Animal Administration and Ethics Committee granted moral approval (No. XMULAC20200148) following the animal welfare tips of the Chinese language Society of Laboratory Animals.
Cytotoxicity analysis
The serum of mice was analyzed for the presence of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (CREA), creatine kinase (CK), and creatine kinase-MB (CK-MB). Following euthanasia, the organs, together with the guts, liver, spleen, lung, and kidney, have been extracted for H&E staining.
Statistical evaluation
The info have been introduced within the type of imply ± SEM. The statistical evaluation employed on this research concerned the utilization of the Scholar’s t-test and one-way ANOVA for evaluating two-group and amongst greater than two teams, respectively. p < 0.05 indicated statistically important variations. * p < 0.05, ** p < 0.01, *** p < 0.001.
