Preparation of tFNAs
The DNA sequences S1, S2, S3, and S4 used to construct the tFNAs were selected based on their demonstrated efficacy in previous studies [11, 14, 15], having been validated to form stable, biocompatible structures capable of effectively penetrating neuronal PC12 cells, reducing oxidative stress, and inhibiting apoptosis. The single-stranded DNAs (ssDNAs) sequences (listed in Table S1) were synthesized by Sangon Biotech (Shanghai, China), and tFNAs were constructed following established protocols [15, 16]. In brief, four ssDNA strands (each at 100 µM) were mixed in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0) and denatured at 95 °C for 10 min. The mixture was then rapidly cooled to 4 °C and maintained at this temperature for 20 min using a T100 thermal cycler (Bio-Rad, CA, USA) to facilitate proper folding and assembly. The successful synthesis and structural integrity of the tFNAs were confirmed using several characterization techniques. Images of tFNAs were obtained with the Benyuan CSPM5500 atomic force microscope (AFM) (Guangzhou, China) after pre-treating the mica plate with poly-L-lysine and incubating a 50 pM tFNAs solution on the mica surface for 30 min. The zeta potential of tFNAs was measured using a Malvern Zetasizer Nano-ZS90 nanoparticle analyzer (Worcestershire, UK) at a concentration of 100 pM in a 1 mL electrode cell. Additionally, polyacrylamide gel electrophoresis (PAGE) was performed to characterize the molecular size of tFNAs.
Cell viability analysis
Cell viability was assessed using CCK-8 reagent (Dojindo Laboratory, Japan). Briefly, cells were seeded into 96-well plates and cultured under conditions specified by the experimental design. Subsequently, 100 µL of CCK-8 reagent was added and incubated for 30 min at 37 ℃. The optical density (OD) at 450 nm was then measured to determine cell viability.
Lipid peroxidation analysis
BODIPY 581/591 C11 (Lipid Peroxidation Sensor) (Invitrogen, USA) was employed to detect ROS in cells seeded in 12-well plates. Cells were incubated with 10 µM Lipid Peroxidation Sensor for 30 min at 37 ℃ in complete growth medium. Following incubation, cells were washed with PBS, and flow cytometry was conducted using FACS analysis (FACScan; BD Biosciences, USA) to capture fluorescent images and quantify the mean fluorescence intensity with FlowJo 10.8 software. Data were collected from a minimum of 20,000 cells.
Measurement of MDA, GSH, and LDH levels
Measurement of MDA
Cellular lipid peroxidation was examined with a Lipid Peroxidation MDA Assay Kit (Beyotime, Shanghai, China). The relative levels were determined by measuring the optical density values at 531 nm using a microplate reader. The concentrations corresponding to each OD value were calculated and expressed in terms of µmol/mg protein.
Measurement of GSH/GSSG ratio
The intracellular levels of GSH and glutathione disulphide (GSSG) were assessed with a GSH Kit (Beyotime, Shanghai, China). The relative levels were analyzed using a microplate reader.
Measurement of LDH
LDH leakage was detected using an LDH cytotoxicity detection kit (Beyotime, Shanghai, China). The relative levels of LDH were quantified by recording the OD values at 490 nm with a microplate reader.
Measurement of iron ions
The intracellular iron concentration was assessed using FerroOrange (Dojindo Laboratory, Japan) following the manufacturer’s protocols. Cells were seeded into dishes and stained with 1 µM FerroOrange for 30 min at 37 ℃. Subsequently, cells were washed with PBS and examined under a confocal microscope. To quantify the Fe2+ levels in brain tissue, the Ferrous Iron Colorimetric Assay Kit (Elabscience, China) was employed. The relative levels were determined by measuring the OD values at 593 nm. The concentrations for each optical density OD were quantified in terms of µmol/kg wet weight.
Western blot
Samples of tissues or cultured cells were collected on ice, then mixed with a RIPA lysis buffer solution (50 mM Tris-HCl, 100 mM NaCl, 1% Triton X-100, 5 mM EDTA, and 1 mM PMSF). This mixture was centrifuged at a speed of 12,000 g for a duration of 15 min. The resulting supernatant was extracted for soluble protein analysis. The protein concentration was determined using the BCA assay method. Proteins from each sample, in equal quantities, were loaded onto 8–15% SDS-PAGE gels for electrophoresis, followed by transfer to 0.45 μm polyvinylidene fluoride membranes (Merck Millipore, USA). The membranes were treated with a 5% nonfat milk solution to prevent non-specific binding, then incubated with primary antibodies as indicated in figure, and subsequently to horseradish peroxidase-linked secondary antibodies. The immunoblots were visualized using an ECL substrate with a luminometer (ChemiScope 6000, Clinx, China), and the signal intensities were quantified with the ImageJ software from the National Institutes of Health (NIH), USA. The list of antibodies used in this study was shown in Table S2.
RNA sequencing
For RNA-sequencing, the three groups of N2a cells (Scramble, Aβ, and Aβ with tFNAs) were harvested and each group contained four replicates. RNA was isolated with Trizol (Invitrogen, CA, USA), and the library was prepared and sequenced with TruSeq PE Cluster Kit (Illumina, MD, USA) on Novaseq platform by Novogene Co., Ltd. (Beijing, China). Clean reads were mapped to mm10 reference genome using Hisat2 (v2.0.5), and the count matrix was obtained with featureCounts (v1.5.0). Differentially expressed genes (DEGs) were identified using DESeq2 (v1.20.0) using the count matrix. Genes with a P-value < 0.05 and absolute value of log (fold change) greater than 0 were assigned as significant DEGs. The normalized FPKM values were used to display the expression level in heatmap.
Animals
The current study was approved by the ethnic committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Both the C57BL/6 background wild type and APP/PS1 mice were maintained with a normal diet on a 12 h light-dark cycle. Intranasal administration of tFNAs was performed for two weeks using a pipette with 10 µL 500 nM tFNAs. Then the behavior tests were performed, and mice were sacrificed.
Optical in vivo imaging
Mice were administered 500 nM of Cy5 or Cy5-labeled tFNAs intranasally. Fluorescence was detected using a multi-mode in vivo imaging system (Model: B Pro, Wuhan BIOVIVO, China). In brief, mice were shaved, imaging was performed using a 200 mm field of view. An excitation wavelength of 630 nm with a bandwidth of 30 nm was chosen to excite the brain, and the emitted light signal was detected at a primary wavelength of 680 nm with a bandwidth of 40 nm. The fluorescence signal exposure time was set to 1 s.
Morris water maze
In the Morris water maze (MWM) experiment, designed to assess spatial learning, mice were trained over a five-day period to locate a submerged platform positioned in a specific quadrant of the maze. Each day, they were subjected to three trials, spaced 30 min apart. For each trial, the mice began from a different quadrant, facing the pool wall, and the trial concluded once the mouse had climbed onto the platform for a minimum of three seconds. If the platform was not discovered within a 60-second timeframe, the mouse was manually directed to the platform and remain there for 15 s. Evaluation of spatial memory was conducted on the seventh day. Throughout the experiment, the mice’s swimming paths, the time taken to locate the platform (referred to as latency), the time spent in the target zone, and their swimming speed were all tracked and analyzed daily. This was achieved using a stationary video camera mounted on the ceiling, positioned two meters above the water surface (Techman, Chengdu, China).
Novel object recognition test
In the novel object recognition (NOR) test, mice were initially acclimated to a 50 cm by 50 cm arena for a duration of 5 min, devoid of any objects, the day before the actual experiment. On the training day, the mice were reintroduced to the arena from a consistent starting point and were given 5 min to explore two distinct objects labeled A and B. Following each exploration session, both the arena and the objects were meticulously cleaned with a 75% ethanol solution to eliminate any olfactory cues. On the test day, object B was removed and replaced with a new object, C. The mice were then allowed another 5 min to investigate both objects A and C. The entire behavior of the mice was captured and analyzed using a video camera that was mounted above the arena (Techman, Chengdu, China). The duration in which the mice interacted with object A was denoted as TA, and the time spent with the novel object C was denoted as TC. The recognition index, an indicator of the mice’s ability to discriminate between familiar and novel objects, was determined using the formula: TC divided by the sum of TA and TC (TC / (TA + TC)).
Golgi staining
Mice were deeply anesthetized with a 2% pentobarbital sodium solution prior to brain extraction. The excised brains were immediately immersed in Solution A/B for 14 days at room temperature. Solution A consists of 5% potassium dichromate and 5% mercury chloride in H2O, and Solution B contains 5% potassium chromate in H2O. Following this, the brains were transferred to Solution C, which was prepared by dissolving 300 g sucrose, 10 g polyvinylpyrrolidone (PVP40), and 300 mL ethylene glycol (C2H6O2) in 500 mL phosphate buffer, and incubated at 4 °C for 7 days. After incubation, the brains were sectioned into 100-micrometer slices using a Vibratome VT1200S (Leica, Germany). Spine imaging of the brain slices was performed using a Nikon Ni-E microscope (Japan). Spine quantification were conducted using ImageJ software (NIH, USA).
Immunofluorescence staining
Mouse brain tissue was first fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned into 3-µm thickness slices. After deparaffinization, the slices were blocked with 3% BSA for 30 min at room temperature before being exposed to primary antibodies (SYP, GLUR2, and NR2B dilution 1:100). The slides were rinsed and then treated with a secondary antibody for an hour at room temperature. Nuclei were stained using the DAPI fluorescent stain. The cells’ fluorescence was observed under a LSM800 confocal microscope from Carl Zeiss in Germany.
Statistical analysis
Statistical analysis was performed using SPSS 26 (IBM, NY, USA). Data were shown as mean ± SD. Two-tailed unpaired t-test was used for single comparisons, and one-way ANOVA was applied for multiple comparisons.
