Supplies
The cyanine 5 (Cy5)-labeled (the cyanine 5 fluorophore hooked up on the 5′-terminus) or bare scramble RNA (siRNA-NC), TP53 siRNA and Trp53 siRNAs (siRNA-Trp53) have been 2′-O-methylated and synthesized by GenScript (Nanjing, China). The reverse complementary sequence of siRNA-TP53 (siRNA-TP53 inhibitor) was synthesized by GenePharma (Shanghai, China). Cisplatin (CDDP) was obtained from TargetMol Chemical compounds, Inc. (Boston, USA). GW4869 was bought from MedChemExpress (MCE, HY-19363). To gather EV-free fetal bovine serum (EV-free FBS) and EV-free plasma, the FBS (Gibco, 10099141 C) and plasma have been centrifuged at 3000 × g for 30 min and filtered by way of 0.45 μm filters (Millipore, HAWP04700), adopted by a second spin at 10,000 × g for 30 min and a last high-speed spin at 100,000 × g for 70 min [32].
Cell tradition and CDDP remedy
Human renal tubular epithelial cells (HK-2 cells) have been bought from the American Kind Tradition Assortment (Rockville, MD, USA) and cultured in DMEM/F-12 medium (DF/12, Gibco, Grand Island, USA) supplemented with 1% antibiotics (penicillin and streptomycin, P/S, Gibco) and 10% EV-free FBS. To induce harm in HK-2 cells, HK-2 cells have been cultured in full DMEM/F-12 supplemented with 10% EV-free FBS and CDDP (the ultimate focus of CDDP was 20 µM) for 12 h. The cells utilized on this experiment have been maintained at 37 °C and 5% CO2.
Platelet isolation
The entire blood of the mice or wholesome human volunteers was collected in EDTA Vacutainer® tubes (BD, Franklin Lakes, USA) and centrifuged at 300 ×g for 20 min. The cell pellet was discarded, and the higher resolution was composed of platelet-rich plasma. The platelet-rich plasma was then additional centrifuged at 3000×g for 15 min, and the ensuing cell precipitate was composed of platelets and erythrocytes. The platelet-free plasma was subsequently fastidiously extracted and saved at 4 °C in a 1.5 mL RNase-free tube. The precipitate was subsequently resuspended in precooled purple blood cell lysis buffer (420301, BioLegend, San Diego, CA) and incubated on ice for five min to eradicate purple blood cells. Afterward, the combination was centrifuged at 3000×g for 10 min to isolate the platelets. The platelets within the precipitate have been resuspended in aseptic regular saline and saved at 4 °C.
Characterization of the nanoparticles fashioned by artificial SiRNAs and plasma
To analyze the nanoparticles fashioned by artificial siRNAs and plasma, 1 ml of EV-free plasma was blended with 0.5 pmol siRNA-Trp53 at room temperature for five min. The combination was then diluted with PBS to scale back the plasma proportion to 10% previous to evaluation by nanoparticle-tracking evaluation (NTA) and transmission electron microscopy (TEM) in accordance with beforehand reported strategies [33]. The zeta potential of the self-assembled siRNA nanoparticles was decided by way of a Zetasizer NanoZS (Malvern Devices, UK).
Mass spectrum
5 milliliters of EV-free plasma was mixed with 2.5 pmol artificial siRNA-Trp53 for 10 min at room temperature. The combination was then diluted with 5 ml of PBS. A management group was established by diluting 5 ml of EV-free plasma with 5 ml of phosphate-buffered saline (PBS). All of the teams have been centrifuged at 120,000×g for two h, after which the sediment was resuspended in 100 µl of sterile PBS for protein mass spectroscopic evaluation by way of LC‒MS/MS.
Preparation of SiRNA (cy5-tagged or bare)-loaded platelets
One milliliter of EV-free plasma was incubated with 0.5 pmol of artificial siRNA-Trp53 (both Cy5-labeled or unlabeled) at room temperature for 10 min to facilitate nanoparticle formation. Subsequent, 108 freshly remoted platelets have been mixed with the nanoparticles and gently rotated to make sure thorough mixing. The combination was then centrifuged at 3,000×g for 10 min. The ensuing pellet was washed twice with PBS to yield platelets loaded with siRNA.
Simultaneous measurement of platelet aggregation and dense granule secretion
The aggregation of platelets and dense granules launch have been assessed concurrently by way of a luminometer (Chrono-log mannequin 700). Aggregation curves have been obtained as a operate of accelerating gentle transmittance by way of stirred PRP samples throughout platelet aggregation. Throughout the identical evaluation, the ATP launch from dense platelet granules from the samples was measured. A high-gain photomultiplier recorded the sunshine emitted throughout the response between platelet-secreted ATP and the firefly luciferin-luciferase combination (chronolume reagent), which was proportional to the quantity of secreted ATP (expressed in nanomoles) following the addition of the stimulator. Aggregation and secretion have been triggered by collagen (2 µg/ml last conc., Chronopar reagent).
Circulate cytometry of the activated platelet marker CD62P and integrin ΑIIbβ3
Human platelets have been stimulated with thrombin (0.01 U/mL). PE-conjugated CD62P (P-selectin) antibody (Invitrogen, 12-0626-82) and GPIIb/IIIa (integrin αIIbβ3) antibody (Abnova, MAB5226) have been used for labeling. The imply fluorescence depth (MFI) was measured by way of movement cytometry (BD Biosciences), and the information have been analyzed by way of FlowJo v10 software program.
Coculture of HK-2 cells and platelets
For confocal fluorescence imaging, 4 × 106 Cy5-labeled siRNA-Trp53-loaded platelets have been incubated with 1 × 104 HK-2 cells in a 12-well plate at room temperature for 12 h. After incubation, the platelets have been eliminated by washing the cells twice with PBS. The HK-2 cells have been then stained with DAPI and imaged by way of laser scanning confocal microscopy. For Western blotting and RT‒qPCR, 4 × 106 platelets loaded with siRNAs have been cocultured with 1 × 104 HK-2 cells. Within the CDDP-treated group, HK-2 cells have been pretreated with 10 nM CDDP for twenty-four h previous to coculture with platelets. Earlier than RNA and protein have been extracted from HK-2 cells for Western blotting and RT‒qPCR, the cells have been rinsed twice with PBS to take away any residual platelets. Equally, for the practical assay within the CDDP-treated group, HK-2 cells have been pretreated with 10 nM CDDP for twenty-four h earlier than being cocultured with platelets. To confirm that Trp53-siRNA was delivered into HK-2 cells by way of EVs, 4 × 106 platelets have been first handled with GW4869 (last focus of 5 µmol/L) for twenty-four h. Subsequently, the platelets have been incubated with nanoparticles fashioned by Trp53 siRNAs within the presence of EV-free plasma for 4 h to gather the pretreated platelets. HK-2 cells (1 × 104) have been handled with or with out CDDP for twenty-four h. Then, the pretreated platelets have been cocultured with HK-2 cells for twenty-four h, and the Trp53 protein ranges have been investigated by way of western blotting, and the cell viability was decided by way of a CCK-8 assay.
Assortment and characterization of platelet-derived EVs
HK-2 cells (5 × 10³) have been initially handled with or with out 20 µM cisplatin (CDDP). Twelve hours later, the CDDP-containing medium was changed with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% EV-free plasma. Twenty-four hours later, 10 mL of the cell tradition medium from the HK-2 cells beforehand handled with or with out 20 µM CDDP was collected and subjected to sequential centrifugation at 3,000×g for 30 min at 4 °C. The samples have been then subjected to centrifugation at 10,000×g for 30 min at 4 °C, adopted by centrifugation at 100,000×g for 70 min at 4 °C in accordance with a beforehand reported protocol [33]. EV-free cell tradition medium was incubated with freshly separated platelets (5 × 106/ml) at 37 °C and 5% CO₂ for 48 h. The medium was subsequently collected and subjected to centrifugation at 3,000×g for 30 min at 4 °C to take away the platelets, 10,000×g for 30 min at 4 °C to take away the cell particles, and 100,000×g for 70 min to gather the EVs launched by the platelets, as beforehand described in a broadcast report [33]. The EVs launched by the platelets have been examined by way of transmission electron microscopy (TEM), nanoparticle monitoring evaluation (NTA), western blotting or RT‒qPCR in accordance with beforehand reported strategies [33].
RNA extraction and reverse transcription quantitative PCR (RT‒qPCR)
For the detection of mRNA, whole RNA was extracted by way of TRIzol™ reagents (Invitrogen, 15596026) in accordance with the producer’s directions. cDNA was subsequently synthesized with the HiScript II 1st Strand cDNA Synthesis Package (Vazyme, R212). Actual-time qPCR (RT‒qPCR) was carried out by way of the ChamQ Common SYBR qPCR Grasp Combine Package (Vazyme, Q711). For siRNA detection, RNA was extracted by way of RNAiso for small RNA reagents (Takara, 9753). A MiRNA 1st Strand cDNA Synthesis Package (Vazyme, MR101) was used for miRNA reverse transcription. cDNA synthesis was carried out for 15 min at 50 °C, and the response was terminated by heating at 85 °C for five min. Actual-time quantitative polymerase chain response (qPCR) was then carried out with the miRNA Common SYBR qPCR Grasp Combine Package (Vazyme, MQ101). Two microliters of cDNA was subjected to 40 cycles of 95 °C for 10 s and 60 °C for 30 s on an ABI 7500 Quick Actual-Time PCR System (Thermo Fisher Scientific). Absolutely the quantitative calculation of siRNA was carried out in accordance with the offered working directions [31]. Briefly, siRNAs (1, 0.5, 0.25, 0.05, or 0.01 pmol) have been dissolved in 1 ml of PBS, and whole RNA was extracted from the combination for qRT‒PCR. A linear curve between the CT worth and absolutely the variety of corresponding siRNAs was drawn to calculate the variety of siRNAs within the resolution. The primers used are listed in Desk S1.
Western blot
The cells and tissues have been harvested with radioimmunoprecipitation assay buffer (RIPA buffer) (R0278, Merck), and the cell particles was depleted after centrifugation at 12,000×g at 4 °C. The supernatant was boiled at 100 °C for five min earlier than being loaded onto a ten% polyacrylamide gel. Proteins resolved by SDS‒PAGE have been transferred onto polyvinylidene fluoride membranes. The membranes have been blocked for one hour and incubated with main antibodies in opposition to Trp53 (1:2000, 2524; Cell Signaling Expertise) and GAPDH (1:5000, G9295; Sigma‒Aldrich) at 4 °C. The membranes have been subsequently washed with Tris-buffered saline and Tween-20 (TBST) and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse IgG, 1:2000, 7076; Cell Signaling Expertise) at room temperature for two h. The luminosity of the protein bands was quantified and analyzed by way of ImageJ software program (Nationwide Institutes of Well being). To establish the markers of platelet-derived EVs, the platelet-derived EVs have been lysed by way of radioimmunoprecipitation assay buffer (RIPA buffer) (R0278, Merck), and western blotting was carried out with anti-CD63 (Cell Signaling Expertise, 10112 S), anti-TSG101 (Proteintech, 28283-1-AP), and anti-ALIX (Abcam, ab117600) antibodies as beforehand reported [32].
Cell counting Package-8 (CCK-8) assay
HK-2 cells (1 × 104) have been handled with or with out CDDP for 12 h. Then, 4 × 106 platelets have been incubated with the HK-2 cells. After 24 h, cell viability was decided with a Cell Counting Package-8 (CCK-8, Yeasen) in accordance with the producer’s directions. To evaluate the protecting impact of siRNA-Trp53-loaded platelet-derived EVs on HK-2 cells in opposition to CDDP-induced toxicity, 8 × 102 HK-2 cells have been seeded in a 96-well plate. Subsequently, the HK-2 cells have been handled with or with out CDDP for 12 h. Then, 0.5 µg (whole protein) of platelet-EVs was incubated with the HK-2 cells. After 24 h, cell viability was decided with a Cell Counting Package-8 (CCK-8, Yeasen) in accordance with the producer’s directions. To establish the protecting impact of siRNA-Trp53 on HK-2 cells derived from platelet-derived EVs, 8 × 10² HK-2 cells have been seeded in a 96-well plate and transfected with a siRNA-Trp53 inhibitor. The HK-2 cells have been then handled with or with out 20 µM CDDP. Following a 12-h incubation interval, 0.5 µg (whole protein) of platelet-derived EVs have been added to the HK-2 cells and incubated for a further 24 h. Cell viability was then decided with a Cell Counting Package-8 (CCK-8, Yeasen) in accordance with the producer’s directions.
In vivo imaging
To show the distribution and efficacy of platelets in delivering medication to injured kidneys, male C57BL/6 mice (8 weeks previous) have been bought from GemPharmatech (Nanjing, China). Wholesome C57BL/6 mice served because the management group. The acute kidney harm C57BL/6 mouse mannequin was induced by ischemia‒reperfusion or CDDP, as beforehand reported [34]. Briefly, for the ischemia‒reperfusion mannequin, 8-week-old male C57BL/6 mice have been anesthetized with isoflurane and positioned on a heated pad to take care of their physique temperature at 37 °C. The bilateral renal vessels have been occluded by way of nontraumatic microaneurysm clamps for 30 min by way of flank incisions. The incisions have been subsequently sutured, and the mice have been returned to their cages. For the CDDP-induced mannequin, CDDP was dissolved in regular saline, sonicated for 20 min, and chilled on ice. All of the mice obtained an intraperitoneal injection of 0.5 mg of CDDP per mouse. Following a two-day interval of ischemia‒reperfusion and CDDP remedy, the mice have been administered Cy5-labeled siRNA-Trp53 or scramble RNA-loaded platelets (5 × 107 per mouse) by way of the tail vein. Twenty-four hours later, the mice have been euthanized and perfused by way of the guts. The mind, coronary heart, lung, liver, kidney, and spleen have been collected. Ex vivo imaging was subsequently carried out to seize and analyze the typical variety of photons per pixel per millisecond of the picture. The renal tissues have been additional sectioned to watch the distribution of the siRNAs and platelets within the renal tissue by way of immunofluorescence staining by way of laser scanning confocal microscopy. An anti-CD41 antibody (ab33661, Abcam) was used to label platelets, and an anti-MCP-1 antibody (ab308522, Abcam) was used to label broken renal tubular cells.
Therapeutic impact of platelets in vivo
To show the therapeutic impact of platelets in delivering medication to injured kidneys, male C57BL/6 mice (8 weeks previous) have been bought from GemPharmatech (Nanjing, China). Two acute kidney harm mouse fashions have been induced by way of ischemia‒reperfusion or CDDP, as beforehand reported [34]. The mice have been randomly assigned to 4 teams: the siRNA-NC group, the siRNA-Trp53 group, the scrambled RNA group, and the siRNA-Trp53 group. The mice have been grouped in line with the injection schedule, with every mouse receiving 0.1 mg of bare siRNA [21] or 107 platelets injected by way of the tail vein each two days, for a complete of 4 injections. Two days after the ultimate injection, the serum was collected from the mice, and so they have been then euthanized to gather the kidneys and livers for western blotting and immunohistochemical staining to judge the therapeutic impact. No less than 5 to 6 mice have been included in every experimental group.
Quantification of BUN, creatinine and neutrophil gelatinase-associated Lipocalin
The urea nitrogen (BUN) ranges have been assessed by way of the DetectX Urea Nitrogen Detection Package from Thermo Fisher Scientific (EIABUN), following the producer’s directions. The serum creatinine ranges have been quantified with a creatinine assay equipment from Nanjing JianCheng Bioengineering Institute (C011-2-1) in accordance with the offered protocol. The focus of neutrophil gelatinase-associated lipocalin within the serum was decided by way of the Mouse NGAL ELISA Package from Proteintech (KE10045) in accordance with the offered handbook.
Tissue histology and immunostaining
The kidneys, livers and hearts have been mounted in 4% paraformaldehyde resolution and embedded in paraffin for hematoxylin and eosin (HE) staining. Renal tubular harm is characterised by tubular dilation, tubular atrophy, tubular solid formation, sloughing of tubular epithelial cells, lack of the comb border, and thickening of the tubular basement membrane. A scoring system was used as follows: a rating of 0 indicated no tubular harm, whereas a rating of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 represented 0–10%, 10–15%, 15–20%, 20–25%, 25–30%, 30–35%, 35–40%, 40–50% and > 50% of tubules injured, respectively. The presence of myocardial harm in every AKI mouse mannequin induced by IRI and CDDP was decided by inspecting the morphology of myocardial cells. For immunohistochemical (IHC) staining, the slides have been incubated with main antibodies in opposition to p53 (10442, Proteintech), Bax (14796, CST), Bcl2 (26593, Proteintech), and cleaved caspase 3 (9661, CST).
Statistical evaluation
Every experiment was repeated a minimal of 3 times. Qualitative information, together with histological pictures, signify findings from no less than three unbiased experiments. All of the statistical analyses have been carried out by way of GraphPad Prism 8 software program, and the outcomes are offered because the means ± normal errors of the means (SEMs). Previous to evaluation, all the information have been assessed for a traditional distribution and equal variance. Relying on the character of the datasets, statistical comparisons have been carried out by way of unpaired Scholar’s t exams and one-way ANOVA. Statistical significance amongst teams was thought-about for values of P < 0.05. For the Western blot experiments, the statistical outcomes are offered after information normalization. This normalization course of was applied to scale back background variations arising from numerous experimental situations. On this examine, significance ranges are denoted by *, **, and ***, representing P < 0.05, P < 0.01, and P < 0.001, respectively.
