Materials
1-Ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), n-hydroxysuccinimide (NHS), sodium citrate, ammonium bicarbonate, were obtained from Sinopharm Holding Chemical Reagent Co Ltd (Shanghai, China). Ammonia, ethanol, methanol, acetone, styrene, and paraformaldehyde were purchased from Sinopharm Holding Chemical Reagent Co Ltd (Shanghai, China). Caffeic acid, triaminophenylboronic acid (APBA) dimethyl sulfoxide (DMSO), and rhizopodophyllin were prepared by Aladdin Chemical Reagent Co. CCK-8 was purchased from Vernon Bio. Reactive Oxygen Assay Kit, Membrane Associated Protein V-FITC/PI Fluorescence Double Staining Apoptosis Assay Kit, Hematoxylin-Eosin/HE Staining Kit were purchased from Beyotime Biotechnology Co. anti-APP, anti-BACE, anti-TNF-α, anti-IL-1β, and anti-IL-10 were purchased from Affinity. anti-β-Tubulin, anti GAPDH were purchased from Proteintech. Mouse IL-1β ELISA Kit, Mouse IL-6 ELISA Kit, Mouse IL-10 ELISA Kit, Mouse TNF-α ELISA Kit, Human IL-1β ELISA Kit, Human IL-6 ELISA Kit Human IL-10 ELISA Kit, Human TNF-α ELISA Kit, Human IL-1β ELISA Kit, Human IL-6 ELISA Kit, Human IL-10 ELISA Kit, Human TNF-α ELISA Kit were purchased from Andy Gene Co. Ltd. Aβ (25–35) was purchased from MedChemExpress Reagents. SH-SY5Y cells and BV2 cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. 5xFAD mice and C57BL/6J mice were purchased from Beijing Huafukang Biological Co.
Preparation of CDs, CDs-CA and CDs-CA-MGs
CDs preparation: CDs containing carboxyl groups were prepared by hydrothermal method. 1.40 g of sodium citrate and 10.5 g of ammonium bicarbonate powder were added to 70 mL of triple-distilled water and stirred until completely dissolved [33]. The reaction was carried out in an autoclave at 180ºC for 8 h, cooled down to room temperature, and then centrifuged at 4ºC and 14,000 rpm for 15 min to remove the precipitate, and the supernatant was taken and used for the subsequent experiments.
CDs-CA preparation: 5mL of CDs solution was added with 0.01 mM EDC and NHS and stirred at room temperature until completely dissolved, then 2 mg of APBA was added and reacted at room temperature for 24 h, and then dialyzed by dialysis (MW = 3500) for 3 days. The solution was changed every 10 h to filter the free APBA. Excessive amount of CA was dissolved into the above solution, and the solution pH was adjusted to alkaline [34]. The solution was stirred for 8 h and then dialyzed with a dialysis bag (MW = 3500) for 3 days, changing the solution every 10 h to remove free CA, and then freeze-drying gave solid powder CDs-CA.
CDs-CA-MGs Preparation: Digestion was performed to collect BV2 cells in which a hypotonic solution was added to rupture the cell membrane. The cell membranes were subsequently ruptured completely using a grinder. Cell membranes were collected by gradient centrifugation. CDs-CA was sonicated with microglial cell membranes at a volume ratio of 1:2 for 30 min, and CDs-CA-MGs was purified by a dextran gel column and freeze-dried to obtain a solid powder [28].
Characterization of CDs, CDs-CA, CDs-CA-MGs
The morphology and structure of the materials were determined by transmission electron microscopy (TEM) (Tecnai G2F30, FEI, USA). The particle size and zeta potential of the synthesized nanoparticles were determined using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Functional groups in CDs-CA-MGs were observed using Fourier infrared spectroscopy (FT-IR, TA, USA).
Brilliant blue staining
The presence of microglial cell membrane proteins in CDs-CA-MGs was qualitatively detected by SDS-PAGE. A certain amount of CDs-CA-MGs and BV2 cell membrane was taken to determine the protein content in the supernatant by BCA. Electrophoresis was performed using SDS-PAGE gels, incubated with 0.25% staining solution for 2 h, decolorized with decolorizing solution for 24 h, and developed.
CDs-CA-MGs drug loading and drug release experiment
The drug content was detected by UV spectrophotometry (UV-2000, Unico, USA) and the drug loading capacity was calculated according to the following formula: drug loading capacity (LC) = (drug mass) / (total weight of nanomaterials) × 100%. In vitro drug release studies were performed in a dissolution apparatus (RC-8D, Tianjin Guoming Medical Equipment Co., Ltd.). The dissolution medium was 1000 ml of phosphate buffer solution with neutral and acidic pH, respectively. The temperature was 37 °C and the paddle speed was 100 rpm. samples (equivalent to 10 mg CA) were placed into the dissolution medium. The dissolution medium (5 mL) was collected at predetermined time intervals for filtration and then supplemented with an equal amount of blank medium. Finally, the concentration of CA in the filtrate was measured by UV (UV-2000, Unico, USA) at 323 nm. All experiments were repeated three times.
Pharmacokinetic and biodistribution in vivo experiment
Male SD rats (200 ± 20 g) of SPF class were selected from the Animal Center of Jinzhou Medical University, Jinzhou, China. 9 rats were randomly divided into 3 groups (n = 3). CA, CDs-CA and CDs-CA-MGs suspension (equivalent to 20 mg/kg CA) were injected intranasally. Orbital venous blood was taken at different time points (0.08 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h, 24 h). Plasma was collected by centrifugation at 8000 rpm for 10 min. The above blood samples were stored at -20 °C to be measured.
When the 5xFAD mice reached 6 months of age, the mice were randomly divided into 2 groups. Mice were injected with CDs-CA and CDs-CA-MGs suspension (equivalent to 20 mg/kg CA) by nasal drip respectively, and brain tissues were collected at different time points (1 h, 2 h, 3 h, 4 h, 5 h, 6 h). Brain tissues of the same weight were weighed and ground into tissue homogenate by adding saline. The supernatant was taken and stored at -20℃ for measurement.
We optimized the research methodology based on the existing [35]. The 200 µL plasma sample or tissue sample was mixed with 10 µL Phloretin (4 µg/mL) and 400 µL methanol. The mixture was vortexed for 3 min, centrifuged at 12,000 rpm for 8 min, and the supernatant was extracted. It was determined by high performance liquid chromatography (HPLC) at a wavelength of 323 nm using methanol and 0.4% phosphoric acid (15:85) as mobile phase, and the metabolic kinetic parameters were calculated by Pksolver 2.0.
CCK-8 experiment
BV2 and SH-SY5Y cells were inoculated in 96-well plates at a density of 10,000 cells/well. An equal volume of CDs-CA-MGs at different concentrations was added to each well (blank and control groups were left). The 96-well plates were incubated in a cell culture incubator with 5% CO2 air at 37 °C for 12 h. 10 µL of CCK-8 solution was added to each well and incubated at 37 °C for 0.5 h. The absorbance at 450 nm was measured by an enzyme marker.
Hemolysis test
Hemolysis test was used to evaluate the in vivo safety of CDs-CA-MGs. Whole blood of mice was taken, centrifuged at 3000 rpm at 4℃ for 10 min, and mixed with saline to configure 2% (V/V) erythrocyte solution. The solution was mixed with different concentrations of CDs-CA-MGs (100, 200, 400, 600, 800, 1000, 1200, 1400 µg/mL) in equal volumes and incubated at 37 °C for 3 h. The absorbance of the supernatant was measured at 576 nm using a microplate reader (Versa Max, Molecular Devices, Sunnyvale, CA, USA). Luminosity. Saline was used as a negative control and deionized water as a positive control. All experiments were repeated three times. Percentage of hemolysis (%) = (sample – negative control)/(positive control – negative control). Hemolysis was considered as hemolysis if the percentage of hemolysis exceeded 5%.
Preparation of Aβ solution
According to the manufacturer’s instructions and relevant literature [1, 36, 37], a stock solution of 1 mg/mL was prepared by adding 1 mL of deionized water to Aβ (25–35) powder. Incubated at 37 °C for 7 days for aging of Aβ monomers to form Aβ oligomers, which were diluted to 10 µM for in vitro experiments. Aβ monomer and Aβ oligomers were co-cultured with certain concentrations of CA, CDs-CA and CDs-CA-MGs, respectively. The structural changes of Aβ were observed using TEM.
Cell culture and model establishment
SH-SY5Y cells and BV2 cells were placed in complete medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin) and incubated in a cell culture incubator at 37℃ with 5% CO2 until the growth stabilization period for cell experiments. Aβ oligomers was diluted to 10 µM and added into the cell culture medium of SH-SY5Y and BV2 cells, to simulate the damaged neurons in Alzheimer’s disease. The cell experiments were categorized into 5 groups of Control, Aβ, CA (Aβ + 10µg/ml CA), CDs-CA (Aβ + 80µg/ml CDs-CA), and CDs-CA-MGs (Aβ + 60μg/ml CDs-CA-MGs) (equivalent to 10ug/ml CA) [38, 39]. Collect the supernatant of BV2 cells and use it as conditioned medium for experiments [40,41,42].
Cellular uptake experiment
The fluorescence intensity of CDs-CA-MGs was measured by confocal laser scanning microscopy to assess the uptake of CDs-CA-MGs by SH-SY5Y and BV2 cells. Briefly, SH-SY5Y and BV2 cells were co-incubated with CDs-CA-MGs solution for 12 h. After fixed staining, the cells were photographed using a confocal laser scanning microscope (CLSM), and the fluorescence intensity of each group was analyzed by ImageJ software.
DCFH-DA experiment
SH-SY5Y and BV2 cells were induced by 10µM Aβ (25–35) for 24 h, then PBS, CA, CDs-CA, CDs-CA-MGs were added and the cells were incubated for 12 h. The cell culture medium was changed, loaded with 10µM DCFH-DA probe, and incubated for 30 min at 37 °C. Cells were washed with PBS for three times to fully remove the DCFH-DA probe. Observe with an inverted microscope.
Flow cytometry
SH-SY5Y, BV2 cells were induced by 10µM Aβ (25–35) for 24 h, then PBS, CA, CDs-CA, CDs-CA-MGs were added and incubated with cells for 12 h. The cell culture medium was changed, loaded with 10µM DCFH-DA probe, and incubated at 37 °C for 30 min. Cells were washed with PBS three times to fully remove the DCFH-DA probe. Digest the treated cells with 0.25% trypsin EDTA, centrifuge, and resuspend the cells with PBS. The cell suspension was placed in a flow assay tube and detected by flow cytometry.
ELISA assay
Collect cell supernatant and tissue homogenate. Standard wells, blank wells, and sample wells were added sequentially to the enzyme marker plate according to the instructions. Then, 100 µL of biotinylated antibody working solution was added to each well and incubated at 37 °C for 1 h. Wash the plate 3 times. Add 100 µL of HRP enzyme conjugate working solution per well and incubate at 37 °C for 30 min. Wash the plate 5 times. Add 90 µL of substrate solution (TMB) to each well and incubate at 37℃ for 15 min, avoiding light. Add 50 µL of termination solution to terminate the reaction. Immediately measure the OD value of each well at 450 nm with an enzyme labeler.The expression levels of IL-1β, IL-6, IL-10, TNF-α and Aβ were calculated in each group according to the instructions.
THT fluorescence intensity detection
Dilute the Aβ monomer solution to 20µM, add a certain concentration of CA, CDs-CA, CDs-CA-MGs solution and mix well. Thioflavin T (THT) was added and incubated at 37℃. The fluorescence signal intensity at the excitation wavelength of 450 nm and the emission wavelength of 485 nm were recorded at 4 time points, i.e., 0, 24, 48 and 72 h, respectively.
Establishment of animal models
5xFAD and C57BL/6 male mice were purchased from the Beijing Huafukang Biotechnology Co. Mouse DNA was extracted by alkaline lysis: 0.1 cm of mouse tail tissue was taken, 100 µL of lysate (25 mmol/L NaOH + EDTA 0.2 mmol/L) was added, and the sample was incubated for 70 min at 90 °C in a metal heated pan. After cooling, an equal amount of 40 mmol of Tris-HCl was added, and then the sample was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant is the desired mouse DNA. The supernatant is the desired mouse DNA solution. PCR amplification was performed using BeyoFusion™ PCR Master Mix (2X). Mouse genotypes were identified by differences in agarose gel electrophoresis bands. 5xFAD mice produce Aβ protein at 1.5 months of age, which rapidly accumulates in the brain, with significant pathological changes at 6 months of age. We selected 6-month-old 5xFAD and C57BL/6 male mice for animal experiments. They were randomly divided into 5 groups (control, AD, CA, CDs-CA and CDs-CA-MGs) with 27 mice in each group. Mice were given CA, CDs-CA, and CDs-CA-MGs suspensions (equivalent to 20 mg/kg CA) intranasally three times daily for 8 weeks [43].
Lipopolysaccharide (LPS)-induced inflammation mouse model: To further investigate the effects of CDs-CA-MGs on inflammation-based cognitive dysfunction. Referring to existing studies [26], we intraperitoneally injected LPS (0.25 mg/kg) for 7 consecutive days to establish a mouse model of LPS-induced cognitive impairment. Mice were simultaneously administered suspensions of CA, CDs-CA, and CDs-CA-MGs (equivalent to 20 mg/kg CA) for 11 days. 3 days later, behavioral tests were performed.
In vivo targeting and uptake experiments
To verify the targeting effect of CDs-CA-MGs, CDs-CA-MGs (20 mg/kg) was injected by nasal drip administration, and brain tissues and hearts, livers, spleens, lungs, and kidneys of mice were taken at set time points. The fluorescence signals in each group of tissues were detected at 518 nm and 494 nm using an in vivo imaging system (IVIS Spectrum, PerkinElmer).
Brain slices were rewarmed at room temperature for 2 h, washed three times with PBS, incubated with 0.3% Triton X-100 for 30 min, and washed three times with PBS. Incubate with goat serum (10-fold dilution of the original solution) for 2 h, incubate with primary antibody at 4℃ for 16 h, wash with PBS for 3 times, incubate with secondary antibody at room temperature for 2 h, wash with PBS for 3 times. DAPI was added dropwise to seal the plate. The fluorescence intensity of each group was analyzed by ImageJ software.
Behavioral experiments
Water maze experiment: Mice were treated for 8 weeks and measured in a water maze with a diameter of 1.2 m. The temperature of the test water was maintained at 22 ± 1 °C. The mice were trained in the water maze. During the training period, a 10 cm platform was fixed at 1 cm underwater. For the first 5 days, mice were trained 4 times at fixed positions in each quadrant of the maze. On day 6, the platform was removed to test the mice’s learning and memory ability. The mice entered the water at a random location in the quadrant away from the platform and swam freely for 60 s. The time to reach the platform, the swimming speed, and the number of times the mice crossed the platform area were recorded.
Nesting experiment: Different groups of mice were moved to cages with the addition of six sheets of paper (6 × 6 cm2). These mice were allowed to move randomly and their nesting behavior was recorded daily by taking pictures and calculating scores.
Nissl staining and HE staining
The specimens were fixed with 4% paraformaldehyde for 48 h, rinsed with ultrapure water for 4 h, then subjected to gradient dehydration, transparency, wax dipping and embedding, and cut into sections of 6 μm thickness. The slices were cut into sections of 6 μm thickness.
Nissl staining: The paraffin sections were routinely deparaffinized to water, and then operated according to the steps of the kit. The sections were stained with methylene blue staining solution for 10 min, differentiated with Nissl differentiation solution for 1 min, and observed under the microscope until the Nissl bodies were clear. Ammonium molybdate solution was treated for 2 min and rinsed with deionized water. Anhydrous ethanol was used for dehydration, xylene was used for transparency, and neutral gum was used for sealing. Observed using an orthogonal microscope.
HE staining: Tissue paraffin sections were routinely deparaffinized to water, following the steps of the kit. The sections were stained with hematoxylin staining solution for 15 min, differentiated with differentiation solution for 30 s, and soaked in tap water for 3 times, each time for 5 min. the sections were put into eosin staining solution for 2 min, and the excess staining solution was thrown off and then dehydrated quickly, and then the sections were transparent in xylene and sealed with neutral gum. The slices were observed with an orthogonal microscope.
Western blotting
The expression levels of inflammatory factors (IL-1β, IL-6, IL-10, TNF-α) and Aβ cleaving enzymes (APP, BACE) were detected in mouse brain tissues by Western blotting. Proteins were extracted from the brain tissues of mice in each group by RIPA lysate (RIPA: PMSF = 100:1). The protein concentration of each group of samples was detected by BCA quantification kit and the samples were prepared. An 8%/10% protein gel was prepared and electrophoresis was performed at a constant voltage of 150 V. Then it was transferred onto PVDF membrane. The membrane was blocked with 5% blocking solution using skimmed milk powder at room temperature for 2 h. After blocking, the primary antibody (anti-IL-1β, 1:1000; anti-IL-6, 1:1000, anti-IL-10, 1:1000, anti-TNF-α, 1:1000; anti-APP, 1:1000; anti-BACE, 1:500; anti-GAPDH, 1:1000; anti-β-Tubulin, 1:20000) was incubated at 4 ℃ on a shaker for 16 h. The primary antibody was recovered and incubated at room temperature on a shaker. The primary antibody was recovered and the secondary antibody was incubated with shaking at room temperature for 2 h. The bands were visualized on a gel imaging system using ECL chemiluminescent agent (A: B = 1:1) and analyzed by ImageJ software.
Immunofluorescence staining
After cell induction treatment, 4% PFA was fixed for 30 min, washed 3 times with PBS. 0.1% Triton was incubated for 15 min. Brain tissue sections were dried at room temperature for 30 min, washed three times with PBS. 0.3% Triton was incubated for 30 min and washed three times with PBS. The sections were blocked with goat serum for 2 h, incubated with primary antibody at 4℃ for 16 h (anti-IL-1β, 1:500; anti-IL-6, 1:500, anti-β-Tubulin, 1:1000). The sections were washed three times with PBS, incubated with secondary antibody at room temperature for 2 h. The sections were washed three times with PBS, and incubated with DAPI for 15 min. The sections were photographed by using an orthogonal microscope, and fluorescence intensities were analyzed in each group by ImageJ software.
Statistical analysis
All data in the experiment were analyzed statistically using GraphPad Prism 8.0 software and expressed as mean ± SD deviation. Comparisons between data from multiple groups were performed using one-way ANOVA. Comparisons between groups were made by LSD-t or Tambane’s analysis; if the variance was not uniform, Kruskal-Wallis test was used. p < 0.05 indicated that the difference was statistically significant.
