Materials
Aβ42 and XD4 peptide was synthesized from Chinese Peptide Company (Hangzhou, China). Both Aβ40 and Aβ42 kits for Aβ measurement were purchased from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan). TNF-α, IL-1β and IL-6 ELISA kits were obtained from Neobioscience Technology Co., Ltd. (Beijing, China). The following antibodies were used: 6E10 (monoclonal raised against Aβ1-16, Signet, SIG39300), W20 (oligomer-specific antibody, developed and prepared in our laboratory), anti-Iba-1 antibody (GenTex, GTX100042), anti-GFAP antibody (Abcam, ab53554), anti-PSD-95 antibody (Abcam, ab18258), anti-synaptophysin antibody (Abcam, ab32127), anti-GAPDH antibody (CST, 2118S), goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Santa Cruz, I1112) or Alexa Fluor 594 (Abcam, ab150084). HRP-conjugated goat anti-mouse or rabbit IgG antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China).
Preparation of W20 and/or XD4 conjugated-SPIONs
W20 and/or XD4 conjugated-SPIONs were synthesized according to previous methods[18]. Briefly, the PEG-coated SPIONs were synthesized by a “one-pot” synthetic approach. 2.1 g of Fe(acac)3, 7.9 mL of oleylamine, and 24 g of HOOC-PEG-COOH (Mn = 2000) were dissolved in 100 mL of diphenyl ether solution and incubated at 80℃ for 4 h with stirring at 400 rpm in anaerobic environment, then the PEG-SPIONs were precipitated by ether and dissolved in PBS for further experiments. After that, 2 mg PEG-SPIONs were mixed with 2.50 μmol EDC and 6.25 μmol sulfo-NHS in 950 μL PBS buffer and incubated for 15 min at room temperature. Then 1 mg W20 and/or 0.1 mg XD4 (in 50 ul of PBS) was added and the reaction was undergoing overnight at 4 °C. The resultant conjugated-SPIONs were collected by centrifugation at 25000 rpm and kept at 4 °C for future use. The conjugative efficiency for W20 or XD4 was calculated by determining the residual protein amount in the supernatant using BCA assay, which was calculated according to the following equation:
Thioflavin T fluorescence assay
To determine the effects of W20/XD4-SPIONs and W20-SPIONs on Aβ aggregation, 10 μM Aβ42 was mixed with 100 μg/ml or 1000 μg/ml of SPIONs and incubated at 37 °C without agitation. When monitoring the aggregation kinetics of Aβ42, a 10 μL aliquot of sample was mixed with 190 μL ThT solution (5 μM), and the ThT fluorescence intensity was measured using a Tecan Safire2 microplate reader (Tecan, Switzerland) set to 450 nm/482 nm (excitation/emission). Data were obtained from three independent experiments.
MTT assay
SH-SY5Y cells (obtained from the cell line resource center of Peking Union Medical College, Chinese Academy of Medical Sciences) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under a 5% CO2 atmosphere. The cells were seeded in 96-well plates with approximately 10000 cells per 100 μL of medium per well. Plates were then incubated at 37 °C for 24 h to allow cells to attach. The SPIONs with or without 4 μM Aβ42 oligomers were added to the wells and then incubated for an additional 48 h at 37 °C. Cell viability was determined by adding 20 μL of 5 mg/mL MTT to each well. After 3 h of incubation at 37 °C, the supernatants were replaced with a 150 μL aliquot of DMSO in the dark. The absorbance at 570/630 nm was measured by using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Data were obtained from three independent experiments.
Measurements for proinflammatory cytokines
BV-2 cells (obtained from the cell line resource center of Peking Union Medical College, Chinese Academy of Medical Sciences) were maintained in DMEM with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2. The cells were treated with the serial of SPIONs with or without 4 μM Aβ42 oligomers and incubated for 12 h at 37 °C. Then the cell supernatants were collected and the levels of TNF-α and IL-6 were determined using ELISA kits (Neobioscience technology, Beijing, China) according to the manufacturer’s protocols. Briefly, the cell supernatants were added to a 96-well ELISA plate and reacted with the relevant primary antibodies followed by HRP-conjugated secondary antibodies. 3,3,5,5-Tetramethylbenzidine was used as the substrate. The absorbance of the samples was measured at 450 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Data were obtained from three independent experiments.
Western blot analysis
Proteins samples from BV-2 cell lysates pretreated with Aβ and a serial of SPIONs were separated by 12% SDS-PAGE gel (Invitrogen) and transferred onto nitrocellulose membrane (Merck Millipore). After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was probed with anti-iNOS (1:1000), anti-COX2 (1:1000) and anti-GAPDH (1:1000) antibodies respectively, and followed by appropriate HPR-conjugated secondary antibodies. Bands in immunoblots were developed with Super-Signal West Pico Plus Chemiluminescent Substrate kit (Pierce, UB278521), and quantified by densitometry using ImageJ software (NIH).
Animal treatment
APPswe/PS1dE9 transgenic mice were obtained from the Jackson Laboratory. All mice were given food and water ad libitum and maintained in a colony room at 22 ± 2℃ with 45% ± 10% humidity under a 12:12 h light/dark cycle. Six-month old male AD mice were categorized into five groups: PBS-treated (n = 8), SPIONs-treated (n = 8), XD4-SPIONs-treated (n = 8), W20-SPIONs-treated (n = 8) and W20/XD4-SPIONs treated (n = 8), and their WT littermates were categorized into three groups: PBS-treated (n = 8), SPIONs-treated (n = 8) and W20/XD4-SPIONs treated (n = 8). The mice were administered with a daily dose of 1 mg nanoparticles in 100 μL PBS (0.01 M, pH 7.4) with 15% mannitol via tail vein for 28 days. After the last administration, the behavioral tests were performed.
Object recognition test
The object recognition test was performed as previously described with slight modifications[30]. Briefly, in the habituation phase, mice were allowed to freely explore the behavioral open-field arena (50 cm × 50 cm × 25 cm white plastic box, empty) individually for 5 min one day before the test was initiated. For the training session (Trial 1), mice were allowed to explore for 5 min in the same box having two identical objects in the upper two corners. For the testing session (Trial 2), after a 24 h retention period, the object in the right corner was replaced with a novel object, and the mice were reintroduced to the box and allowed to explore for 5 min. Time spent exploring and sniffing each object was recorded. The results are expressed as the discrimination index by calculating: . The box was cleaned with 70% alcohol between tests to eliminate olfactory cues.
Y-maze test
The Y-maze test consisted of 2 trials separated by an interval of 1 h. The first trial was 10 min in duration and allowed the mouse to explore only two arms (the start and familiar arms) of the maze, with the third arm (novel arm) blocked. In the second trial, the mouse was put in the same starting arm as in trial 1 with free access to all 3 arms for 5 min. The total time spent and the number of entries in the novel arm were video recorded and analyzed. The arms were cleaned with 70% alcohol between trials to eliminate olfactory cues.
Morris water maze test
The water maze consisted of a pool (110 cm in diameter) containing opaque water (22 ± 1 °C) and a platform (10 cm in diameter) submerged 1 cm under the water. Hidden platform training was carried out twice per day over five consecutive days, with an inter-trial interval of 3-4 h. Mice were allowed to swim for 60 s to find the platform, on which they were allowed to stay for 10 s. The trial ended when the mouse located the platform. Mice unable to locate the platform were guided to it. 24 h after the acquisition trial, the mice were tested for memory retention in a probe trial in the absence of the hidden platform. The performance of each mouse was monitored using a video camera (Sony, Tokyo, Japan) mounted over the maze and automatically recorded via a video tracking system.
Immunohistochemistry
Mice were deeply anaesthetized with avertin (250 mg/kg) and transcardially perfused with ice-old PBS containing heparin (10 U/mL) before sacrificed. Their brains were immediately removed and divided along the sagittal plane. The left brain hemisphere was fixed in 4% paraformaldehyde at 4 °C overnight and processed for paraffin-embedded sections. Coronal paraffin-embedded serial sections of 5 μm thickness were cut on a Lecia CM1850 microtome (Leica Biosystems, Buffalo Grove, IL, USA). For immunohistochemistry analysis, sections were deparaffinized and subjected to antigen retrieval using citrate buffer (0.01M, pH 6.0, 0.05% Tween-20) in a steamer at 95 °C for 20 minutes. The sections were then incubated with 3% H2O2 to quench endogenous peroxidases and washed 3 times with 1×TBS. Thereafter, sections were permeabilized and blocked with 10% normal goat serum in 0.3% Triton X-100 PBST for 1 h at room temperature. Subsequently, the sections were incubated with 6E10 (1:100), anti-Iba-1 (1:100) and anti-GFAP (1:100) antibodies, respectively, followed by corresponding HRP-labeled secondary antibody and visualized with diaminobenzidine (DAB). For synaptophysin and PSD-95 staining, sections were immunostained with anti-synaptophysin (1:100) and anti-PSD95 (1:100) antibodies followed by corresponding secondary antibody conjugated to Alexa Fluor 488 (1:200) or Alexa Fluor 594 (1:200). All images were acquired with an Olympus IX73 inverted microscope with DP80 camera. For immunostaining of 6E10, Iba-1 and GFAP, the positively stained area in at least three sections from cortex and striatum per mouse were analyzed, and three fields for each section were imaged and quantified. For immunostaining of PSD-95 and synaptophysin, immunofluorescence intensities were quantified. All images were processed and analyzed by ImageJ Software (National Institutes of Health, USA).
For microglia engulfment analysis, paraffin-embedded sections of 20 μm thickness were immunostained for Aβ with 4G8 (1:100) and microglia marker with anti-Iba-1 antibody (1:100) followed by corresponding secondary antibody conjugated to Alexa Fluor 594 (1:200) and Alexa Fluor 488 (1:200), respectively. The brain sections were imaged on a Leica TCS SP8 confocal microscope. The Aβ puncta engulfed by the Iba-1-positive microglia were quantified. 8-10 microglia cells were analyzed per mouse.
Brain lysate preparation
The right brain hemisphere was homogenized in RIPA buffer containing protease inhibitor cocktail (Sigma, P2714-1BTL). The tissues were then centrifuged at 14,000 × g for 30 min at 4 °C, and the supernatant (RIPA-soluble fraction) containing soluble Aβ was collected. The pellets were resuspended in guanidine buffer (5.0 M guanidine-HCl/50 mM Tris-HCl, pH 8.0) and centrifuged at 14,000 × g for 1 h at 4 °C to obtain supernatants containing insoluble Aβ (guanidine-soluble fraction).
Measurements for Aβ and proinflammatory cytokines in the brain lysates
The levels of RIPA-soluble Aβ and RIPA-insoluble (guanidine-soluble) Aβ in the brain lysates of mice were quantified by ELISA using Aβ40 and Aβ42 immunoassay kits (Immuno-Biological Laboratories). For the proinflammatory cytokine measurements, the levels of TNF-α, IL-1β and IL-6 in the brain lysates of mice were determined using corresponding ELISA kits (Neobioscience Technology) according to the manufacturer’s protocols.
Measurement of GSH, GSSG and ROS
The levels of GSH and GSSG in the brain lysates were assayed by commercial kits (Beyotime, S0053). Total GSH was measured by 5, 5-dithiobis (2-nitrobenzoic) acid (DTNB)-GSSG reductase recycling. GSSG was obtained by determining the absorbance of 5-thio-2-nitrobenzoic acid produced from the reaction of the reduced GSH with DTNB according to the manufacturer’s protocols. The reduced GSH was obtained by subtracting GSSG from the total GSH. The absorbance was determined at 412 nm by using an MD-M5 microplate reader.
ROS assay was performed as previously described[31]. Briefly, ROS production was fluorometrically monitored using 2, 7-dichlorofluorescein diacetate (DCFDA) mixed with the brain lysates. The intensity of DCF fluorescence generated from carboxy-DCFDA was proportional to the amount of ROS. The fluorescence was determined using an MD-M5 microplate reader (excitation, 485 nm; emission, 530 nm) and the ROS units were calculated.
Statistical analysis
Data were analyzed with GraphPad Prism v.8. Statistical significance was assessed using the student’s t-test, one-way or two-way ANOVA followed by Bonferroni post hoc test, as appropriate. Results were expressed as group mean ± SEM, and P < 0.05 was considered statistically significant. All samples or animals were included for statistical analysis unless otherwise noted in pre-established criteria.