Materials and reagents: PLGA (mol wt 20,000, 50:50 dl-lactide:glycolide copolymer) was purchased from Beijing Haosail Technology (Beijing, China). L-CS (mol wt 50,000, 85% deacetylated) was purchased from Haidebei Marine Bioengineering (Jinan, Shandong, China). M-HA (M-HA; mol wt 80,000–100,000) was purchased from Heowns Biochemical Technology (Tianjin, China). Triserine (N-terminal cysteine, Cys; purity ≥ 90%) was synthesized and purchased from ChinaPeptides (QYAOBIO, Shanghai, China). The pcDNA3.1-BMP7 (pDNA-BMP7) and pcDNA-3.1-EGFP (pDNA-EGFP) overexpression plasmids were custom designed in-house and outsourced for synthesis by GenScript (Nanjing, Jiangsu, China). All chemical reagents were purchased from Sigma-Aldrich (Merck, Beijing, China), unless otherwise stated. All tissue culture reagents were from HyClone (Thermo Fisher Scientific, Beijing, China) and plasticware was from BD Biosciences (San Jose, CA), unless otherwise stated. TGF-β1 was purchased from R&D Systems (Shanghai, China).
Layer-by-Layer Assembly and Nanoparticle Characterization
Firstly, PLGA nanoparticles (P-NP) were prepared using the water-in-oil-in-water (w/o/w) double emulsion-solvent evaporation method. PLGA (10 µg mL-1) was dissolved in dichloromethane to form the organic phase. Ultra-pure water was used as the first aqueous phase and was added to the organic phase. A pre-emulsion was obtained by ultrasonication of the solution in an ice-cold water bath. A second aqueous phase containing 8% v/v polyvinyl alcohol (PVA, mol wt 30,000–70,000) was added to the pre-emulsion and a secondary emulsion (water-in-oil, w/o) was obtained by ultrasonication in an ice-cold water bath. Then, the w/o emulsion was added to 1% v/v PVA to form a w/o/w emulsion following an additional ultrasonication step in an ice-cold water bath. The w/o/w emulsion was stirred at room temperature for 5 h to volatilize the organic solvent, and then the solution was centrifuged (14,000 rpm, 10 min) to collect solid P-NP. The P-NP were washed three times with distilled water to remove residual surfactant. The synthesis of Cy5@P-NP utilized the same method but a concentration of 0.1 mg mL-1 Cy5 (DBCO-Cy5, Sigma-Aldrich) was dispersed in the ultra-pure water.
The P-NP were alternately transferred to pre-filtered 0.1 м sodium acetate buffer solutions containing L-CS (0.6 mg mL-1, 0.5 mol L-1 NaCl), pcDNA3.1 plasmids (50 ng µL-1), and M-HA (0.1 mg mL-1, 0.5 mol L-1 NaCl) for 30 min per step and under constant stirring at 1,000 rpm. NP were separated from each solution following centrifugation (8,000 rpm, 10 min) before transfer to the subsequent solution. Each step in the three-step process was repeated a total of three times to achieve LbL assembly and adsorption through electrostatic interaction. The LbL assembled NP were centrifuged at atmospheric pressure for 10 min before rinsing three times with distilled water to yield PCH-NP. Alternatively, the final M-HA layer was replaced with a HS layer to yield PCHS-NP.
The dry-state morphologies of the prepared NP were determined using TEM (JEM-2800, JEOL, Tokyo, Japan). Samples were diluted with Milli-Q water and then dropped onto a copper wire, dried at room temperature, and negatively dyed with phosphotungstic acid. The hydrodynamic diameters and polydispersity indices of the prepared NP were measured using DLS (NanoBrook ZetaPlus, Brookhaven Instruments, Holtsville, New York, US). The zeta potential measurements of the prepared NP were acquired using laser Doppler electrophoresis (Zetasizer Nano ZS90, Malvern Panalytical, Malvern, UK) at a constant temperature (25°C, RT). Samples were dispered in salt-free Milli-Q water and transferred to DTS1070 folded capillary cuvettes (Malvern) prior to analysis. Each sample was measured in triplicate and across a minimum of 10 runs.
Cell Culture: Human kidney mesangial cells (passages 4–8) were isolated from donor tissue according to previously detailed methodology,[88] identified according to previously detailed methodology,[88, 89] and kindly provided by Prof. Xueyuan Bai, National Clinical Research Center for Kidney Diseases, Beijing, China. Human PTECs (HK-2; passages 5–15) were purchased from China Center for Type Culture Collection (#GDC0152, Beijing, China). Human PTECs and mesangial cells were seeded in cell culture plates and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies, Carlsbad, California, US) mixed 1:1 with Ham’s F12 (Gibco) containing 10% (v/v) FBS (Gibco) and 1% (v/v) antibiotic − antimycotic solution (HyClone). All cells were cultivated at 37°C in a humidified atmosphere containing 5% CO2.
Transfection efficiency
Cells were grown to 50% confluency on Nunc Thermanox coverslips (Thermo Fisher Scientific), in six-well culture plates before treatment with 10 mg mL-1 PCH-NP or PCHS-NP encapsulating 2% w/w pDNA-EGFP. After 48 h, cells were washed, fixed with 4% paraformaldehyde, and mounted in DAPI Fluoromount-G (ThermoFisher Scientific). Cell expression of EGFP was visualized using a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany). Cells expressing EGFP were counted, and the percentage efficiency of transfection was determined. Naked pDNA-EGFP (equivalent concentration) with no carrier served as negative controls. Lipofectamine LTX (Invitrogen, Thermo Fisher Scientific) transfection reagent encapsulating equivalent concentrations of pDNA-EGFP served as positive controls. Alternatively, transfected cells were detached from culture plates and resuspended in PBS to a density of 1 x 106 cells mL-1 and immediately assessed for positive EGFP/FITC expression using a flow cytometer (LSRFortessa, BD Biosciences). Data were analyzed using FlowJo v10 software (Tree Star, San Carlos, CA).
Reverse transcription and quantitative real-time PCR (RT-qPCR)
Total RNA was isolated from cells or homogenized tissues using RNeasy Mini Kits (Qiagen Sciences, Germantown, Maryland, US), according to the manufacturer’s protocols. A total mass of 1 µg RNA was reverse transcribed into cDNA using PrimeScript RT Kits (Takara Biomedical Technology, Beijing, China), according to the manufacturer’s protocols. The RT-qPCR reactions were prepared using Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) according to the manufacturer’s protocols, and the analyses were performed using a StepOnePlus Real-Time PCR System (Applied Biosystem, Thermo Fisher Scientific). Triplicate test reactions were carried out for each sample to analyze gene expression, which was normalized to the house-keeping gene, GADPH. Relative expression of mRNA was calculated according to the 2−ΔΔCt method. Negative cDNA controls were cycled and assessed in parallel with each run. The custom primers used in RT-qPCR experiments are provided in Table S1.
Western Blot Analysis
Total protein was extracted from the lysates of cells and homogenized tissues after incubation and agitation in RIPA lysis buffer containing protease inhibitors (Solarbio, Beijing, China). A total mass of 25 µg total protein extract from each sample was loaded into the wells of 10% SDS-PAGE gels for separation by electrophoresis at 120 V for 90 min. The separated proteins transferred to polyvinylidene fluoride (PDVF) membranes at 100 V for 90 min in pH 8.3 buffer. Target proteins were detected by standard immunoblotting procedures. Briefly, membranes were blocked with 5% skimmed milk/0.1% Tween-20/PBS for 90 min at RT, followed by incubation with primary antibodies diluted in 5% skimmed milk/0.1% Tween-20/PBS, overnight at 4°C. Then, following wash steps, membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody in 5% skimmed milk/0.1% Tween-20/PBS for 120 min at RT and in darkness. Protein bands were detected following incubation of membranes with ECL reagent and exposure on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Relative densitometries were calculated using ImageJ v1.53t-Fiji (NIH, Bethesda, Maryland, US). The antibodies and dilutions used for Western blot analyses are provided in Table S2.
Transwell migration assays
Transwell migration assays were performed as previously described.[90] Briefly, a total volume of 200 µL mesangial cells or PTECs suspension (1 × 105 mL-1) were seeded in the upper chambers, and 600 µL culture media was added to the lower chambers of 24-well Transwell culture plates. After 12 h, the media of lower chambers were substituted with serum-free media in the presence or absence of 10 ng mL-1 TGF-β1 and/or 10 µg mL-1 NP preparations. Following a further 24 h of culture, cells were rinsed with PBS and fixed with 4% paraformaldehyde for 30 min. Migrated cells were identified in the lower chambers by staining with 0.1% crystal violet for 30 min followed by gentle washes with PBS. Images of the migrated cells were taken using a camera attachment for optical microscopy (DM3000; Leica, Wetzlar, Germany) and subsequently quantified.
Immunocytochemistry
Mesangial cell or PTECs were grown on 24-well cell slides to 50% confluency before culture with serum-free medium in the presence or absence of 10 ng mL-1 TGF-β1 and/or 10 µg mL-1 NP preparations. After a further 48 h of culture, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. The cells were then permeated with 0.1% Triton X-100 for 10 min at RT, blocked with 1% goat serum for 45 min, incubated with primary antibodies overnight at 4°C, and then co-stained with secondary antibodies in the dark at RT. Finally, the nuclei were labeled with DAPI. Confocal images were obtained using a confocal laser microscope (LSM880, Carl Zeiss, Germany). ImageJ v1.53t-Fiji (NIH) was used to determine the mean fluorescent intensities. The antibodies, reagents, and dilutions used for immunocytochemistry are provided in Table S2.
Oxidative stress injury cell models
PTECs were seeded into 12-well plates and incubated with the indicated concentration of H2O2 (Thermo Fisher Scientific) in the presence or absence of M-HA, triserine, or HS for 12 h. After three gentle washes with PBS, cell viability was assessed using CCK-8 assays (company/country) or the production of superoxide by mitochondria was determined following incubation of cells with 5 µm MitoSOX Red mitochondrial superoxide indicator (Yeasen) for 10 min. MitoSOX+ cells across 6 microscopic fields of view were counted, averaged, and presented as a percentage of the total cells, as indicated by DAPI staining.
Animal studies and renal fibrosis models
A total of 77 C57BL/6 mice (male, 8–10 weeks old, weight 22–24 g) purchased from Beijing Vital River Laboratory Animal Technology (Charles River, Beijing, China) were used in this study. All animal experiments were approved by the Animal Experiments Ethical Committee of Nankai University and complied with the NIH Guide for Care and Use of Laboratory Animals. Mice were acclimatized for 2 weeks before experiments and were housed in standard rodent cages with ad libitum access to water and food. Cages were arranged to allow equivalent exposure to light during the day-night cycles. A total of 27 mice were used for in vivo NP biodistribution analyses.
For the UUO model, mice were randomly assigned into groups of 5 for Sham, UUO, P-NP, and PCHS-NP treatments. Mice were anesthetized by isoflurane inhalation, ventral flank fur was removed with depilatory cream, and the skin was sterilized with ethanol. A small 2-cm incision through the skin and peritoneal cavity was made and the ureter of the animal’s left kidney was tied off using a 6–0 silk suture. The peritoneum and skin were then sutured using medical-grade sutures. Animals were kept at 37°C and monitored until they regained consciousness. On the third day post-UUO surgery, mice received either 100 µL saline, or 100 µL saline containing 25 mg kg-1 body mass of P-NP or PCHS-NP by tail vein i.v. injection. At 7 days post-UUO surgery, animals were euthanized by isoflurane inhalation followed by overdose injection of chloral hydrate. Blood was cleared by perfusion injections of PBS into the left ventricle of the heart prior to organ collection. Major organs, and both the UUO kidney and healthy kidney were harvested for further evaluation.
For the FA model, mice were randomly assigned into groups of 6 (n = 12 individual kidneys) for Saline, FA, EI, LI, and RI treatments. Mice were intraperitoneally (i.p.) injected with saline containing folic acid (FA) (200 mg kg-1 body mass) or saline alone (Saline group). Every 7 days mice received either 100 µL saline, or 100 µL saline containing PCHS-NP (25 mg kg-1 body mass) by tail vein i.v. injection, according to the specified treatment parameters for each group. At 28 days post-FA i.p. injection, animals were euthanized by isoflurane inhalation followed by overdose injection of chloral hydrate. Blood was collected for assessment of SCr and BUN, and the remaining blood was cleared by perfusion injections of PBS into the left ventricle of the heart prior to organ collection. Major organs and both kidneys were harvested for further evaluation.
In vivo biodistribution of NPs
A volume of 100 µL saline containing 25 mg kg-1 body mass of Cy5@P-NP, Cy5@PCH-NP, or Cy5@PCHS-NP were administered to healthy mice by tail vein i.v. injection. The major organs of the mice were harvested at 4, 12, and 24 h post-injection. Ex vivo imaging and image-based signal intensity quantification of Cy5 in the kidneys were performed with the use of a NightOWL II LB 983 in vivo imaging system (IVIS) and the IndiGO software package (Berthold China Ltd., Beijing, China), respectively.
Histopathology, immunohistochemistry, and immunofluorescence staining
Kidney tissue was halved and fixed in 10% formalin overnight, following gradient dehydration, kidney samples were embedded in paraffin, cut to 6-µm thick sections, and mounted on glass microscopy slides. Sections were stained with hematoxylin and eosin (H&E) to observe tissue architecture and tubular atrophy; periodic acid–Schiff (PAS) was used to observe hyaline cast formation. Masson’s trichrome was used to visualize deposition of collagen.
For immunohistochemistry, formalin-fixed, paraffin-embedded kidney tissue sections were deparaffinized and stained using conventional primary and secondary-HRP antibody procedures with diaminobenzidine (DAB) as a chromogen for visualization of HRP activity.
For immunofluorescence staining, kidney tissue was halved and fixed in 10% formalin overnight and then embedded in OCT compound (Tissue Tek; Sakura Finetek, Torrance, CA) for cryo-sectioning to 4-µm-thick sections for immunofluorescence staining. Immunostaining was performed to visualize proliferating cells (Ki67), pro-fibrogenic cells (α-SMA), healthy tubules (LRP2, megalin), mesangial cells (PDGFRα), and macrophages (CD68, iNOS), following the antibody and appropriate secondary antibody application instructions recommended by the manufacturer (Abcam). Staining was quantified using ImageJ v.1.53t-Fiji software (NIH). Fully blinded tissue collection and sectioning (three separate researchers), staining and imaging (three separate researchers), and data analysis (two separate researchers) were performed by different researchers throughout the investigation. Details of the antibodies used are included in Table S2.
Hydroxyproline Assay
Hydroxyproline, a component of collagen, was measured in the kidney tissue homogenates using the Hydroxyproline Assay Kit (Solarbio), according to the manufacturer’s protocols. Briefly, kidney tissue homogenates were mixed with 6 mol L-1 HCl and hydrolyzed for 6 h at 110°C. After cooling the mixture, the pH was adjusted to 6–8 with NaOH, then the mixture was centrifuged at 16,000 rpm for 20 min. The supernatant was collected, incubated with the kit reagents, and analyzed on a microplate reader at a 560 nm detection wavelength. Hydroxyproline standards were prepared alongside and used to calculate the absolute hydroxyproline content.
DHE Staining of Tissue ROS
Frozen kidney sections in OCT were prepared for DHE staining (Sigma-Aldrich), according to the manufacturer’s instructions. Briefly, slides were rinsed once in PBS before being immediately immersed in 1 µм DHE staining solution. After incubation for 30 min at RT, the slides were immersed in PBS for three 5 min washes. Confocal microscopy (LSM880, Carl Zeiss, Germany) was used for imaging, and ImageJ v.1.53t-Fiji software (NIH) was used to quantify the mean fluorescence intensity of the DHE fluorescence.
Statistical Analysis
Quantitative data from experimental replicates are presented as mean ± s.d. Quantitative data obtained from averaged datasets are presented as mean ± s.e.m. Data were analyzed using GraphPad Prism (v8 software). No statistical outliers were identified and thus, no datapoints were excluded in this study. Statistical differences between two independent data groups were assessed by a two-tailed independent Student's t-test. Statistical differences between univariable multiple data groups or bivariate multiple data groups were evaluated using one-way ANOVA with Tukey’s or Dunnett's post hoc test or two-way ANOVA with Tukey's post hoc test, respectively. Data with significant differences are marked as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ns = no significance. All statistical tests are indicated in the associated figure legends and statistical indicators are annotated on the relevant figure panels.