Polyglycerol‐Amine Covered Nanosheets Target Cell‐Free DNA to Attenuate Acute Kidney Injury

Abstract Increased levels of circulating cell‐free DNA (cfDNA) are associated with poor clinical outcomes in patients with acute kidney injury (AKI). Scavenging cfDNA by nanomaterials is regarded as a promising remedy for cfDNA‐associated diseases, but a nanomaterial‐based cfDNA scavenging strategy has not yet been reported for AKI treatment. Herein, polyglycerol‐amine (PGA)‐covered MoS2 nanosheets with suitable size are synthesized to bind negatively charged cfDNA in vitro, in vivo and ex vivo models. The nanosheets exhibit higher cfDNA binding capacity than polymer PGA and PGA‐based nanospheres owing to the flexibility and crimpability of their 2D backbone. Moreover, with low cytotoxicity and mild protein adsorption, the nanosheets effectively reduced serum cfDNA levels and predominantly accumulated in the kidneys to inhibit the formation of neutrophil extracellular traps and renal inflammation, thereby alleviating both lipopolysaccharide and ischemia‐reperfusion induced AKI in mice. Further, they decreased the serum cfDNA levels in samples from AKI patients. Thus, PGA‐covered MoS2 nanosheets can serve as a potent cfDNA scavenger for treating AKI and other cfDNA‐associated diseases. In addition, this work demonstrates the pivotal feature of a 2D sheet‐like structure in the development of the cfDNA scavenger, which can provide a new insight into the future design of nanoplatforms for modulating inflammation.


Experimental details
Equipment. NMR spectra were recorded with a Jeol Eclipse (Jeol, Japan) nuclear magnetic resonance spectrometer (500 MHz). UV-visible absorption spectra were measured using a U-3310 spectrophotometer (Hitachi, Japan). Zeta potential and dynamic light scattering were conducted in phosphate-buffered saline (PBS) with a pH of 7.4 (Malvern NANO ZSPO, UK).
Transmission electron microscopy (TEM) imaging was performed with a FEI Tecnai G2 F30 TEM. cfDNA assays were measured with a multi-well plate reader (Bio-Tek, Winooski, USA).
Patient samples: AKI was identified and classified according to the kidney disease improving global outcomes (KDIGO) criteria. [1] The characteristics of the AKI patients (n = 46) from our hospital are described in Supplemental Table S2. Eleven of these patients had biopsy-proven AKI. Healthy volunteers (n = 47) with matching sex and age were selected as controls, and kidney tissues (n = 6) were taken from donor kidneys that were deemed unsuitable for transplantation. Blood samples were collected within 72 hours after AKI diagnosis, and those enrolled AKI patients received conservative management without dialysis, including underling cause of kidney disease, life-threatening complications, nephrotoxic medications, etc. The study was approved by the First Affiliated Hospital of Sun Yat-Sen University Institutional Review Board (Guangzhou, China). All patients and healthy volunteers provided their written informed consent.
Human hemocyte isolation: Venous blood samples from healthy volunteers was drawn into ethylenediaminetetraacetic acid tubes. Human peripheral platelets were isolated and purified using a platelet isolation kit (Solarbio, China) following the manufacturer's protocol. Briefly, blood samples were diluted with an equal volume of PBS and added to the surface of the separation solution. After centrifugation at 1000g for 20 minutes, the platelet-rich plasma layer was retained and washed with PBS until the purified platelets were obtained. Human peripheral blood neutrophils were isolated and purified using a MACSxpress® neutrophil isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's protocol.
Hemolysis activity and biocompatibility assessment: The in vitro hemolysis assay was conducted on human erythrocytes. The hemolytic activity of M-PGA-M and PGA was examined at various concentrations in the range of 1-100 µg/mL, with water and PBS as the positive and negative controls, respectively. Cells were incubated on a shaker at 37 °C for 3 hours, followed by centrifugation at 4 °C (3000 rpm, 5 minutes). Supernatants were read at 540 nm in a Multiskan FC microplate reader. Results were expressed as percentages of the control.
The morphology of the erythrocytes was observed with a microscope (Olympus, Japan). For the cytocompatibility assay, either 100 µg/mL M-PGA-M or PGA was added to a mixture of human platelets and neutrophils (at a ratio of 50:1) and incubated at 37 °C with 5% CO2 for 4 hours. [2] Cells were fixed, stained with a Wright-Giemsa stain (LEAGENG, China), [3] and observed under a microscope.
Cell culture and treatment: HK-2 cells (American Type Culture Collection) were cultured in DMEM medium with 10% FBS until the cells were 80% confluent. Cells were treated with either LPS (20 µg/ml), LPS with M-PGA-M, or LPS with PGA (2-4 µg/mL) at 37 °C with 5% CO2 for 12 hours. Then, the medium was replaced with serum-free DMEM to culture for another 12 hours. The supernatants from various groups were used to measure the cfDNA or collected as a conditioned medium. The conditioned medium was added to the mixtures of platelets and neutrophils from healthy volunteers and incubated at 37 °C with 5% CO2 for 4 hours. Then, the mixed cells were used for immunofluorescent staining of platelets and NETs. Quantification of NET formation: NET formation was quantified by detecting DNA release spectrophotometrically with the DNA-binding dye SYTOX Green, as previously described. [4,5] The mixed cells were stained with 500 nM SYTOX Green and the fluorescence intensity was The combination of LPS and CpG (LPS-CpG) induced AKI model in mice was performed and modified from previously described. [7] Briefly, mice were intraperitoneally injected with For ischemia reperfusion injury, renal pedicles were clamped for 30 min, as previously reported. [8] Sham-operated mice were performed abdominal incision, but without renal pedicle clamping. For treatment, a single-dose of M-PGA-M (10 mg/kg) or PGA (10 mg/kg) was given though intravenous injection 1 hour before modeling. Their littermates injected with saline were sat as controls. Animals were sacrificed at 24 hours.
Biochemistry index and renal histology: Blood samples were collected to determine serum creatinine, blood urea nitrogen, aspartate transaminase, alanine transaminase, creatine kinase and creatine kinase-MB using a Toshiba Automatic Biochemistry Analyzer (Toshiba, Tokyo, Japan). Serum cfDNA was detected using a Quant-iT™ PicoGreen™ dsDNA Assay Kit.
Kidney histology was examined on paraffin-embedded sections stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). To evaluate the tubular injury score, at least 10 random tissue sections per animal were assessed from the PAS staining in a blinded manner by two renal pathologists. Tubular injury was defined as tubular dilatation, tubular atrophy, loss of the brush border, formation of tubular casts and interstitial edema, [9] and semi-quantified according to the area of the tubular lesion as follows: score 0, no tubular damage; score 1, up to 25%; score 2, 25%-50%; score 3, 50%-75%; score 4, more than 75%. Data are the results from at least six mice per group. followed by incubation with secondary antibodies. [10] Nuclei were counterstained with DAPI.
Images were captured using a Zeiss LSM 880 microscope with Airyscan confocal superresolution. The percentage of positive staining was analyzed and quantified with ImageJ software.
Oxidative stress assay: The mixtures of platelets (200 × 10 6 /mL) and neutrophils (4 × 10 6 /mL) cultured in confocal dishes were treated with corresponding the conditioned medium at 37 °C with 5% CO2 for 4 hours. Cells or frozen mouse kidney sections were fixed, permeabilized and blocked. The generation of ROS was detected using a DCFH-DA assay (Dojindo, Japan) [2] according to the manufacturer's protocol. Super-resolution images were obtained using a Zeiss                     Tukey's multiple comparison test (*P < 0.05, compared with Sham; ＃ P < 0.05, compared with IRI).