Sodium tetrachloropalladate (II) (Na2PdCl4, > 98%) was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Insulin (bovine pancreatic, 27 u/mg), sodium hydroxide (NaOH), hydrochloric acid (HCl, 36–38%) and nitric acid (HNO3, 65–68%) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). A superoxide anion assay kit, hydroxyl radical assay kit and H2O2 detection kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A Cell Counting Kit-8 (CCK-8) assay kit was obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China), and an ROS assay kit was purchased from Abcam Inc. (ab238535). PBS was obtained from Procell Life Science and Technology Co., Ltd. (Wuhan, China). Ultrapure water (18.2 MΩ cm− 1) was purified with a Milli-Q system.
Synthesis of Pd@insulin clusters
Pd@insulin clusters were prepared by a biomimetic insulin incubation method. Insulin powder (30 mg) was routinely dissolved in 50 mL deionized water at 37 °C with magnetic stirring, and 25 µL Na2PdCl4 solution (100 mM) was added gradually. Subsequently, the pH of the solution was adjusted to 10–11 with 1 M NaOH. After 24 h, a pellucid Pd@insulin cluster solution was obtained and dialyzed (Mw = 3 kDa) against ultrapure water. A Pd@insulin cluster powder was obtained by freeze drying.
Characterization of Pd@insulin clusters
TEM and high-resolution transmission electron microscopy (HRTEM) were conducted with a JEM-2100 microscope operated at 200 kV and a JEM-2100 microscope equipped with an EDX energy-dispersive spectrometer. SEM was performed with a Hitachi S4800 microscope operated at 3 kV. XPS was conducted on a PHI-5000 C ESCA system (Perkin Elmer) with Mg Kα radiation using an internal standard (C1s peak at 284.6 eV). CD was measured by a Thermo Scientific Nicolet iS10 spectrometer in the range of 4000–400 cm− 1 and a Jasco J-815 spectrophotometer. Size and stabilization were evaluated by dynamic light scattering (DLS, Nano-ZS90, Malvern) and ultraviolet-visible (UV-vis) spectrophotometry (Nanodrop 2000, Thermo Fisher). TGA was conducted by Mettler Toledo TGA/DSC3+. The ion concentration was measured by inductively coupled plasma mass spectrometry (ICP-MS, iCAP RQ, Thermo Fisher).
ROS-scavenging ability of Pd@insulin clusters in solution
The H2O2, O2*− and OH* scavenging capacities of the clusters were determined by assay kits. H2O2-scavenging capacity was evaluated by reacting H2O2 with ammonium molybdate, which resulted in the formation a yellow solution with an absorbance peak at 405 nm. The concentrations of superoxide anion, which was generated by the reaction between xanthine and xanthine oxidase, and hydroxyl radical, which was produced by the Fenton reaction, were determined at a wavelength of 550 nm via chromogenic Griess reagent. EPR was performed to determine the free radical (superoxide anion and hydroxyl radical)-scavenging ability of the Pd@insulin clusters. hydroxyl radical, which was generated from H2O2 by UV-laser irradiation, was captured by BMPO, while superoxide anion, which was generated by the reaction between 2.5 mM KO2 and 3.5 mM 18-crown-6, was captured by DMPO. The magnetic field signals of hydroxyl radical and superoxide anion were detected by spectrometry (Bruker A300, Germany).
Density functional theory (DFT) calculation
The palladium cell was cleaved (220) crystal plane to build (3 * 3 * 1) supercell, and the Pd (220) supercell of five original layers were fixed in the next two layers and relaxed the rest of the atomic layers. All the self-consistent periodic DFT calculations were carried out using the DMol3 code as implemented in the Materials Studio package. The electron exchange and correlation were described with GGA-PBE functional, while the DFT-D (G06) method was used in DFT calculations for the dispersion correction. The localized double-numerical quality basis set with a polarization d-function (DNP-3.5 file) was chosen to expand the wave functions. The core electrons of the metal atoms were treated using the effective core potentials (ECP), and the orbital cutoff were 4.0 Å for all atoms. Brillouin-zone integrations were performed on a k-point mesh sampling grid of 3*2*1 by Monkhorst-Pack with a thermal smearing of 0.008 Ha. For the geometry optimization, the convergences of the energy, Max. force, and Ma. displacement was set as 1 × 10 − 5 Ha, 2 * 10 − 3 Ha/Å, and 5 * 10 − 3 Å, and the SCF convergence for each electronic energy was set as 1.0 * 10–6 Ha. All the transition states (TSs) of the elementary reactions are identified using a complete linear synchronous transit and quadratic synchronous transit (LST/QST) approach. The convergence criterion was set for the root-mean-square forces on the atoms to be 0.01 Ha/Å.
Three cell lines, i.e., N2a, BV2 and A172 cells, were used. N2a and BV2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) with 1% streptomycin and penicillin in a 37 °C incubator, whereas A172 cells were cultured in DMEM/F12 containing the same concentrations of FBS and antibiotics. Primary microglia were cultured in DMEM containing 10 ng/mL GM-CSF, 10% FBS, 1% streptomycin and penicillin.
In vitro cluster uptake
N2a, BV2 and A172 cells were seeded into 24-well plates. After 24 h, Cy3 (Cy3-NHS, GLPBIO)-labelled Pd@insulin (Pd@insulin-Cy3) was added to the cells for 1, 2, 4, 6 or 8 h, and the cluster uptake efficiency of each cell type was assessed with a Zeiss AX10 fluorescence microscope and ZEN 2.3 (blue edition) software.
ROS-scavenging ability of Pd@insulin clusters in vitro
A commercial ROS assay kit consisting of a DCFH-DA probe and Rosup (a positive control) was employed to evaluate the in vitro ROS-scavenging capacity of Pd@insulin. N2a, BV2 and A172 cells were seeded in 24-well plates, and high ROS levels were induced by Rosup (50 µg/mL, 20 min) treatment. Then, the medium was replaced with Pd@insulin or insulin solution for 2 h. Afterwards, the cells were washed with serum-free medium three times to remove the free clusters/insulin, and fresh medium containing 10 µM DCFH-DA probe was added for 30 min. Finally, the cells were washed three times with serum-free medium for fluorescence imaging.
Evaluation of the biocompatibility of Pd@insulin clusters in vitro
The cytotoxicity of the clusters was determined by the CCK-8 assay. N2a, BV2 and A172 cells were seeded in 96-well plates at a density of 104 cells per well and incubated at 37 °C and 5% CO2. After 24 h of incubation, the medium was removed and replaced with fresh culture medium containing different concentrations of Pd@insulin/insulin (Additional file 1: Table S2). After 1 or 2 days of incubation, the culture medium was replaced with CCK-8 solution (10 µL per 100 µL medium), and the cells were incubated for 30 min. Then, cell viability was quantified by measuring the absorbance at 450 nm with a microplate reader (Versa Max, Molecular Devices).
C57BL/6J mice were housed and bred at the Comparative Medicine Animal Facilities of Tongji University School of Medicine. All procedures were conducted according to protocols approved by the Institutional Animal Care and Use Committee of Tongji University School of Medicine (reference number SYXK (HU) 2014-0026).
Analysis of blood glucose levels in mice
Fresh blood (5 µL) was obtained from the tail of each mouse, and blood glucose levels were measured with a commercial glucometer (Yuyue 580, Jiangsu).
Evaluation of the biocompatibility of Pd@insulin clusters in vivo
Eight- to ten-week-old male mice were treated with 250 µL Pd@insulin for 6 successive days (intravenously). ECG was conducted to evaluate mouse heart function (Labchart, ADInstruments). Major tissues, including heart, liver, spleen, lung, kidney and brain tissues, were isolated for H&E staining, and blood samples were collected for blood panel analysis and serum biochemistry tests, i.e., analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALB, TBil, urea and Cre levels. The presence of occult blood and transferrin in excreted faeces was assessed with test strips.
Depression- and anxiety-like behaviors were assessed by the TST, FST, and SPT. Eight- to ten-week-old male mice were treated with 250 µL Pd@insulin for 6 successive days (intravenously). On day 7, each mouse was suspended by the tail with its head 25 cm away from a table using adhesive tape for 6 min, and the immobility time in the final 4 min was recorded with a camera. In the FST, the mice were placed in a glass cylinder (height: 30 cm, diameter: 20 cm) filled with water (23–25 °C) to a depth of 15 cm for 6 min, and immobility time in the final 4 min was recorded. To explore the sucrose preference of cluster-treated mice, all mice were exposed to two bottles of water, two bottles of 2% sucrose solution, and water deprivation for 24 h in advance. Finally, each mouse was given one bottle of water and one other bottle of 2% sucrose solution, and sucrose preference was calculated by determining the ratio of water consumed to sucrose solution consumed.
BBB-crossing ability of Pd@insulin clusters
To explore the ability of the clusters to cross the BBB, Cy5-NHS (APExBIO) was used to label Pd@insulin (Pd@insulin-Cy5) and insulin (insulin-Cy5). Pd@insulin clusters (1 mL) were incubated with Cy5-NHS (1 mg/mL) for 1 h at room temperature. Extra free dyes were removed by repetitive ultrafiltration centrifugations to obtain labeled Pd@insulin clusters. After intravenous injection, the distribution of the labeled Pd@insulin clusters throughout the bodies of the mice was recorded at different time points by an in vivo fluorescence imaging system (VISQUE In vivo Smart-LF, Vieworks), and the distribution in brain tissue was assessed. To further prove that the clusters were able to cross the BBB, the Pd2+ ion concentration was measured by ICP-MS, and brain sections from cluster-treated mice were subjected to TEM and EDX analysis and immunohistochemical staining (DAPI, 1:1000; MAP2, mouse, cat# MB0078, Bioworld, 1:100; Iba1, goat, cat# ab5076, Abcam, 1:100; GFAP, chicken, cat# AB5541, Millipore, 1:500).
Pharmacokinetics and biodistribution of Pd@insulin clusters
The pharmacokinetics of Pd@insulin was assessed by measuring the Pd2+ ion concentration in mouse blood and analyzed by a two-compartment pharmacokinetic model. At different time points (1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 24, and 48 h), fresh blood (5 µL) was obtained from the tails of Pd@insulin-treated mice and dissolved in aqua regia for quantification by ICP-MS. The biodistribution of the clusters was quantified by determining the Pd2+ concentration in tissue. To avoid interference by blood, Pd@insulin-treated mice were perfused with PBS, and major tissues, including heart, liver, spleen, lung, kidney and brain tissues, were isolated. To assess metabolism, the feces and urine of cluster-treated mice were collected at different time points (8 h, 24 h, 48 and 72 h). Finally, major tissues, feces and urine were dissolved in aqua regia for 2 days and passed through a 0.22 μm filter. The Pd2+ concentration in the obtained solution was determined by ICP-MS. Kidney and intestine sections were observed by TEM and EDX analysis.
Severe controlled cortical impact (CCI) injury was induced in 8- to 10-week-old male mice. The mice were routinely anaesthetized with 4% chloral hydrate and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA). After sterilization, the scalp was retracted, and a 4 mm craniotomy centered between the lambda and bregma sutures was performed. The skull pieces were carefully discarded, taking care to prevent disruption of the underlying dura disruption. Subsequently, the angle between the impacting piston and exposed cortex was corrected, and the mice underwent impact (deformation: 1.2 mm; piston velocity: 3.05 m/sec) (Impact One TM Stereotaxic Impactor for CCI, Leica Microsystem). Sham mice underwent anesthesia but did not undergo CCI.
Treatment of TBI mice
The mice were randomly divided into the naïve (control), sham and TBI by CCI groups. After surgery, the TBI mice were randomly divided into two groups: a group intravenously injected with 250 µL Pd@insulin cluster solution for 6 days and a group treated with the same volume of PBS.
ROS-scavenging ability of Pd@insulin clusters in TBI mice
Brain tissues were isolated from euthanized mice, minced for 30 min, and digested with 2.5% trypsin, and a single-cell suspension was obtained by passing the tissues through filters twice (70 μm and 40 μm). According to the protocol of the assay kit, catalyst and DCFH solution were successively mixed with the single-cell solution. After 30 min and three rinses in serum-free medium, the fluorescent ROS signal was analyzed by flow cytometry (BD-LSRFortessa, BD).
Balance beam test The motor function of the mice was evaluated by the balance beam test. The mice were subjected to training 3 days before CCI, and the test was performed on days 3, 7 and 14 after TBI. Each mouse was placed on the starting point on a wooden beam (width: 0.5 cm, length: 100 cm) elevated 60 cm above the ground. On the training days, the mice were trained to cross the beam and enter a closed black box spontaneously. If a mouse failed to cross the beam, it was gently guided forward. On the testing days, the average time to cross the beam, total step number, and missed step ratio were recorded manually.
Y maze test The Y maze consisted of 3 grey glass arms (length: 30.5 cm, width: 9 cm, height: 14.5 cm) at a 120° angle to each other. The mice were habituated to two arms for 6 min, during which the other arm was blocked. On the testing day, the blocked arm (defined as the novel arm) was opened, and the time spent the novel arm (in the final 4 min of a 6-min period) was recorded for each mouse with a video camera.
Barnes maze test A circular acrylic plastic table (diameter: 115 cm) with 18 holes (diameter: 7 cm) spaced equidistant around the perimeter was used. Training was performed for 4 days, during which each mouse was placed in the center of the maze and covered with an opaque bowl for 10 s before being allowed to explore the maze and locate and enter an escape box beneath the table in the presence of noise interference. The latency to enter the target zone was recorded. If a mouse failed to find the escape box within 5 min, it was gently guided to enter the target. On the testing day, the escape box was replaced, and the tendency of the mice to explore the primary escape box was evaluated by measuring the number of entries into the target quadrant and distance travelled in the target quadrant.
Brain sections were fixed in 4% paraformaldehyde, washed three times with PBS, and incubated in permeabilization and blocking buffer (3% BSA, 10% donkey serum, and 1% Triton X-100 in PBS) for 1 h. The brain sections were incubated with primary antibodies including GFAP (chicken, cat# AB5541, Milipore, 1:500), Iba1 (goat, cat# ab5076, Abcam, 1:100), NeuN (mouse, cat# MAB377, Sigma-Aldrich, 1:500), DCX (rabbit, cat# 4604, Cell Signaling Technology, 1:800), Sox2 (mouse, cat# 4900s, Cell Signaling Technology, 1:200), TNFα (rabbit, cat# ab183218, Abcam, 1:500), IL6 (rabbit, cat# 66146-1, Proteintech, 1:50), BDNF (rabbit, cat# ab108319, Abcam, 1:500), and cleaved-caspase3 (rabbit, Cell Signaling Technology, cat# 9664s, 1:400) overnight at 4 °C. Then, all sections were washed with PBS and incubated with secondary antibody for 1 h. Immunofluorescence was observed under a confocal microscopy (FV3000, Olympus).
Mouse brain sections were washed twice with PBS and treated with a mixture of labelling solution and enzyme solution (In Situ Cell Death Detection Kit, TMR red, Sigma) for 1 h at 37 °C; sections not treated with enzyme solution were used as negative controls. Then, the sections were washed twice with PBS and observed by confocal microscopy.
Mice were randomly divided into the sham, TBI and TBI + Pd@insulin groups and sacrificed for brain tissue collection after 6 days of treatment. Total RNA was extracted using a mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and samples with an RNA integrity number (RIN) ≥ 7 were used for subsequent analysis. Libraries were constructed using a TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. These libraries were sequenced on the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X Ten), and 125-bp/150-bp paired-end reads were generated.
Transcriptome sequencing and analysis were conducted by OE Biotech Co Ltd. (Shanghai, China). Raw data (raw reads) were processed using Trimmomatic. Reads containing poly N and low-quality reads were removed to obtain clean reads. Then, the clean reads were mapped to the reference genome using HISAT.
The FPKM and read counts value of each transcript (protein-coding) were calculated using bowtie2 and eXpress. DEGs were identified using the DESeq (2012) functions estimateSizeFactors and nbinomTest. A P value < 0.05 and a fold change > 1.5 or fold change < 0.5 were set as the thresholds for significantly different expression. Hierarchical cluster analysis of DEGs was performed to assess transcript expression patterns. GO enrichment analysis and KEGG pathway analysis of the DEGs were performed using R based on hypergeometric distribution.
PPIs were analyzed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) algorithm.
Quantitative real-time polymerase chain reaction (qRT-PCR)
cDNA was generated from mRNA using OligodT primers with a Transcriptor First Strand cDNA Synthesis Kit (HiScript III All-in-one RT SuperMix Perfect for qRT-PCR, Vazyme). An RNase inhibitor was used to prevent degradation. Amplification was performed using Taq Pro Universal SYBR qRT-PCR Master Mix (Vazyme) and specific primer sets (Supplementary Table 3). mRNA expression levels were normalized by to the expression of GAPDH.
The concentrations of TNF-α (abs520010, ABSIN) and IL-6 (KMC0021, Invitrogen) in the microglial culture medium were measured following the manufacturer’s protocol.
Protein was extracted from microglia using M-PER Protein Extraction Buffer (Pierce) containing protease inhibitor cocktail (Sigma). The protein concentration was determined via the BCA method. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred on to polyvinylidene fluoride membranes (Millipore and Bio-Rad). The membranes were incubated with primary antibodies against COX2 (rabbit, cat# ab15191, Abcam, 1:1000), CD68 (rabbit, cat# ab125212, Abcam, 1:1000), β-actin (mouse, cat# a5441, Sigma, 1:5000), and TNFα (rabbit, cat# ab183218, Abcam, 1:1000) overnight and then incubated with a horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technologies, 1:5000). The bands were visualized with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, United States).
Differences between two independent groups were analyzed by unpaired Student’s t-test, and differences between multiple groups were analyzed by one-way/two-way ANOVA followed by Tukey’s post hoc test. The data are shown as the mean ± SD, and P < 0.05 was considered significant.