A40-PO polymersome preparation and characterization
P[(OEG)10MA]20-PDPA120 and angiopep2-P[(OEG)10MA]20-PDPA120 copolymers were synthesized via atom transfer radical polymerization (ATRP) as previously reported.33 A40-POs were formulated adhering to the film-rehydration method, whereby P[(OEG)10MA]20-PDPA120 and a 1.88% molar ratio of angiopep2-P[(OEG)10MA]20-PDPA120 were dissolved in a mixture of methanol and chloroform (v/v, 3:1). The mixture was left to evaporate, allowing the organic solvent to completely dissipate and form a homogeneous polymer film at the bottom of the vial. PBS solution was added to rehydrate the polymer film and sonicated for 30 min. The solution was stirred continuously for 7 days at 4 °C via a magnetic stirrer at 1000 rpm. The morphology of the A40-POs was characterized via transmission electron microscopy (JEM-2100Plus, Japan). The diameter distribution was assessed via dynamic light scattering (Malvern Zetasizer Pro, UK).
Animals
All the animal studies were conducted in accordance with the guidelines set forth by the West China Hospital Animal Care Committee (IACUC-approved project number: 20211475A). Considerable efforts were made to minimize the number of animals utilized in these studies and to alleviate any pain or discomfort experienced by the animals. In all the experiments, the animals were housed in a room where the temperature was regulated, with consistent alternating cycles of light and darkness.
Immunohistochemistry (IHC)
Paraffin-embedded sections (5 μm) of mouse brain tissue were deparaffinized with xylene and subjected to gradient alcohol hydration (100%, 80%, 50%, 30%). Endogenous peroxidase activity was quenched via the addition of 3% H2O2 (room temperature, 10 min) following antigen retrieval (Biosharp, 22315828). The tissues were blocked with 1% BSA (Aladdin, A104912) at room temperature for 30 min. Aβ-targeting primary antibodies were incubated overnight at 4 °C (Servicebio, GB13414-1, 1:200), followed by washing with PBS. The sections were then incubated with HRP-conjugated secondary antibodies (ABclonal, AS014, 1:150) at ambient temperature for 30 min and subsequently washed with PBS. DAB substrate (Elabscience, E-IR-R101, 1:20) was applied for 5 min, followed by hematoxylin solution (Servicebio, G1004) for 1 min and hematoxylin differentiation solution (Servicebio, G1039) for 10−15 s. The sections were immediately rinsed in running tap water for 20 s, treated with hematoxylin bluing buffer (Servicebio, G1040) for 1 min, and washed with PBS. Finally, the slides were mounted with quick-drying neutral resin (ZSZSGBBIO, ZLI-9516).
Aβ extraction
Following euthanasia via an overdose of isoflurane anesthesia, cardiac perfusion was conducted with cold PBS. The entire brain tissue was subsequently harvested and weighed. The brain tissues were subjected to comprehensive homogenization (Servicebio, KZ-5F-3D) employing a TBS solution enriched with phosphatase inhibitor (Servicebio, CR2302054) and protease inhibitor (Servicebio, CR2306008) at 70 Hz and −20 °C. The supernatant was segregated after the brain tissue homogenate was centrifuged for 1 h at 100,000 × g and 4 °C via an ultrahigh-speed centrifuge (Beckman, Optima MAX-XP). The sediment were subsequently resuspended in 70% formic acid (Aopusheng (Tianjin) Chemical, 20210610) and centrifuged at 100,000 × g and 4 °C for 1 h, after which the resulting supernatant was collected.
Vessel extraction
Immediately following euthanasia via isoflurane overdose anesthesia, cardiac perfusion was conducted with cold PBS, then brain tissues were excised and weighed. The tissues were processed via vascular parenchyma isolation buffer comprising 10 mM HEPES (Servicebio, CR2207064), 141 mM NaCl (Aladdin, 111549), 4 mM KCl (Aladdin, P112134), 2.8 mM CaCl2 (Aladdin, C290953), 1 mM MgSO4 (Aladdin, M433513), 1 mM NaH2PO4 (Aladdin, S433623), and 10 mM glucose (Servicebio, CR2112094). To 500 μL of brain tissue homogenate, 1 mL of 26% (w/w) dextran (Next Sage, 61212ES60) was added. Following thorough mixing and a resting period of 15 min at 4 °C, the mixture was centrifuged (15,800 × g, 15 min, 4 °C). The upper layer subsequently represented the parenchymal fraction, and the lower layer corresponded to the vascular fraction. The vascular fraction was washed with PBS 2 × 5 min for further detection.
Enzyme-linked immunosorbent assay (ELISA)
The parenchyma and blood vessels underwent comprehensive homogenization using a tissue lysis mixture (Yase, 016c1050) enriched with a phosphatase inhibitor (Servicebio, CR2302054) and a protease inhibitor (Servicebio, CR2306008) at 70 Hz and −20 °C. The tissues were allowed to fully lyse at 4 °C for 15 min. The supernatant was collected via centrifugation at 14,000 × g and 4 °C for 10 min. The protein concentrations of various proteins, including LRP1 (NOVUS, NBP3-00449), PACSIN2 (Anruike, YX-160103M), Rab5 (abbexa, abx154598), and Aβ (Invitrogen, KMB3441), were determined via ELISA kits following the manufacturer’s protocols. For Aβ in the blood, the test was performed directly according to the ELISA instructions. Aβ extracted by formic acid needs to be neutralized with Tris base before testing. Protein concentrations were calculated on the basis of the curve equation (four-parameter fit).
Confocal imaging
Confocal images were captured via a Leica Stellaris 5 confocal microscope equipped with Diode 405, Argon, DPSS 561, and HeNe633 lasers. Imaging was conducted at a resolution of 2048 × 2048 pixels and a scanning speed of ×100. Colocalization analysis to derive PCC (r) was performed via the colocalization plug-in for ImageJ.
STED imaging
STED nanoscopy experiments were performed under a Leica DMi8 confocal microscope equipped with a Leica TCS SP8 STED-ONE uni. The dyes (TSA570, TSA620 and TSA750) were excited under an STED laser. The emission signals were collected via HyD reflected light detectors. The depletion beam was applied at wavelengths of 592 nm, 660 nm, and 775 nm (50% power), with a resolution of 2048 × 2048 pixels and a scanning speed of ×100.
Tyramide signal amplification (TSA) stain
Mouse brain tissue sections were deparaffinized with xylene and hydrated through gradient alcohol dehydration. Following antigen retrieval, 3% H2O2 was utilized to inactivate endogenous peroxidase (Biosarp, 22315828) for 10 min. Permeabilization of the sections at room temperature for 10 min was achieved with 1% Triton X-100 (Biofroxx, 1139ML100). The sections were rinsed with PBS and incubated with the primary antibody for 1 h at room temperature. After washing with PBS, the samples were incubated with the HRP-conjugated secondary antibody for 10 min. Subsequent to a PBS rinse, the TSA fluorescent solution (Absin, abs50031) was applied and incubated at room temperature for 10 min. Antigen retrieval was subsequently conducted. The aforementioned steps were repeated with distinct primary antibodies, including antibodies against LRP1 (ABclonal, A1439, 1:100), Aβ (Servicebio, GB13414-1, 1:200), CD31 (Cell Signaling, 77699, 1:300), Rab5 (Thermo Fisher, PA5-88260, 1:200) and CD146 (Abcam, ab75769, 1:300). Various wavelengths of TSA, including the TSA520, TSA570, TSA620, and TSA700 dyes, until multimeric fluorescence staining was achieved. The nuclei were stained with DAPI (Solarbio, C0065) for 5 min and then sealed with antiquenching sealing agent (Solarbio, S2100).
Positron emission tomography-computed tomography (PET-CT) imaging
APP/PS1 POs (12-month-old) mice were injected intravenously with the commercial Aβ radiocontrast agent [18F] AV-45 (2.8–3.2 MBq). Then, intracranial images of the mice were acquired via a micro-PET-CT imager (Inviscan, France) according to operation specifications. After a recovery period, saline (200 μL) was intravenously injected into wild-type mice (3- and 12-month-old) and sham APP/PS1 mice (12 months old). The APP/PS1 POs were intravenously injected with 200 μL of A40-POs (10 g/L). After 12 h, all the mice were intravenously injected with [18F] AV-45 (2.8–3.2 MBq). Intracranial images of the mice were acquired via the same imager.
Hematoxylin‒eosin (H&E) staining
Mice tissue sections (5 μm) were dewaxed and rehydrated. The sections were stained with hematoxylin solution for 5 min, immediately rinsed in running tap water for 10–15 s, followed by incubation with hematoxylin differentiation solution (Servicebio, G1039) for 10–15 s. Subsequently, the sections were treated with hematoxylin bluing solution (Servicebio, G1040) for 30 s and washed in running tap water. The sections were then stained with eosin staining solution for 30 s, followed by washing with ethanol. The slides were dehydrated through a gradient of ethanol (100%, 80%, 50%, 30%) and xylene. Seal the sections with quick-drying neutral resin (ZSZSGBBIO, ZLI-9516).
Tissue clearing and staining
The paraformaldehyde-fixed mouse brain were treated with 1/2 CUBIC-L solution (80 mL Milli-Q, 5 g Triton X-100, and 5 g N-butyldiethanolamine (Aladdin, 102-79-4)) at 37 °C for 6 h. Then, the solution was replaced with a CUBIC-L solution and incubated at 37 °C for 15 days, with the new CUBIC-L solution being replaced every 2 days. The brain tissues were subsequently washed three times with staining buffer (1.5 M NaCl). The hyalinized brains were transferred to staining buffer containing a vasculature probe (Vector, DL-1178-1, 1:100) and an Aβ probe (Abcam, ab216983, 100 nM) for fluorescence staining (RT, 3 days). The refractive index was adjusted via a CUBIC-M solution (25 g Milli-Q, 45 g antipyrine (Aladdin, 60-80-0), 30 g N-methylnicotinamide (Aladdin, 114-33-0), and 125 μl of N-butyldiethanolamine)). Images were analyzed via Amira and iMaris software.
High-performance liquid chromatography (HPLC)
Donepezil HCl (Aladdin, D129948) was dissolved and added to a polymer film preparing donepezil HCl@A40-POs, followed by dialysis using a 3 kDa dialysis bag for 7 days. The concentration of donepezil HCl in the dialysis fluid was measured to calculate the encapsulation efficiency. Sodium 1-decanesulfonate (Aladdin, S100284) was dissolved in pure water (15.7416 mM/L) and filtered. Chromatographic grade acetonitrile solution and perchloric acid were added, followed by ultrasonication for 10 min. Standard solutions of donepezil HCl at concentrations of 250, 125, 62.5, 31.25, 15.625, and 7.8125 μg/mL were prepared. The content of donepezil HCl was detected via an Agilent-1260 chromatograph at a flow rate of 1 mL/min, a column temperature of 35 °C, a volume of 20 μl, and a detection signal at 271 nm.
Morris water maze (MWM) experiment
In each group, the mice were administered a caudal vein injection daily (A40-POs, donepezil@A40-POs, donepezil, or saline), which was continued for three days. The mice were subsequently housed in a standard rearing environment for seven days to acclimatize and recover. For the analysis, the pool was segmented into four quadrants. From days 11 to 14 (days 375–378 of lifespan), a platform was positioned in quadrant II, and the animals were introduced into the thermostatic pool from each quadrant daily, with time taken by the mice to locate the platform recorded as escape latency. If the mice failed to reach the platform within 120 s, they were guided to it and remained there for 30 s. A spatial probe (Stage II) was performed on day 15 (day 379 of lifespan). Once the platform was removed, the mice were placed in the water from the IV quadrant. The results of the spatial probe were expressed as either the percentage of time the mice remained at the original escape platform location or the number of times they passed. Reverse place navigation trials were conducted from days 16 to 19 (day 380–383 of lifespan), with the platform positioned in the IV quadrant which opposite the original platform location. The animals repeated the regimen from days 11 to 14, and the reverse escape latency was documented. On day 20 (day 384 of lifespan), the platform was removed, and the mice were introduced into the water from quadrant II. The results of the reverse spatial probe are expressed as either the percentage of time that the animal spent on the original platform position or the number of times it crossed the location. In addition, 6 months later, the mice were subjected to place navigation (stage V) and spatial probe (stage VI) experiments again with the same methods and conditions. Animal performance was recorded by the same tracking system (EthoVision XT, Noldus Information Technology) for each stage.
Sucrose preference
At the end of Stage IV and Stage VI, the sucrose preference of the sham, APP/PS1 and APP/PS1 POs was tested. Each mouse was housed individually and allowed to acclimatize to the cage, which contained two bottles of standard pure water, for 2 days. One of the bottles was subsequently filled with 2% sucrose solution. The amount of water consumed by the mice was recorded, and the water in each bottle was refreshed daily. Sucrose preference = Sucrose water consumption/(Sucrose water consumption + Standard purified water consumption) × 100%
Nest construction
Three days after the sucrose preference experiment, each mouse was housed individually and then adapted to a single-cage environment for 7 days. Subsequently, 10 pieces of paper (5 × 5 cm2) were added to each cage and evenly placed. After 4 days, the nesting test results were scored in accordance with the improved 4-point scoring system.57 1 point, no visible tear, no recognizable nest site; 2 points, no visible tear, nest site recognizable; 3 points, partial tear, recognizable nest site; 4 points, sharpest tear, recognizable nest.
Digital western blot
Western blot analysis was performed via the Jess automated digital system (ProteinSimple, Santa Clara, CA, USA) with 12–230 kDa separation modules. Total protein concentrations were determined via a bicinchoninic acid (BCA) assay (Biosharp, BL520A). The samples were diluted in 1× sample buffer and 5× master mix (ProteinSimple) to a final loading concentration of 1 μg/μL, denatured at 95 °C for 5 min. They were then loaded into wells at a volume of 3 μL per well. Denatured samples, blocking buffer, primary antibodies, HRP-conjugated secondary antibodies (Jess modular antibodies, ProteinSimple), wash buffer, and chemiluminescent substrate (1:1 luminol-peroxidase mixture) were sequentially loaded into the designated detection plate wells. The primary antibodies used included rabbit anti-CD31 (1:50; Cell Signaling Technology, #77699), anti-CD146 (1:50; Abmart, T55209), anti-NeuN (1:50; Abcam, ab177487), and anti-β-actin (1:300; Servicebio) antibodies. Automated capillary electrophoresis, immunoblotting, and signal acquisition were performed via the Jess system according to operation specifications. The data were analyzed with Compass for Simple Western software (v6.2, ProteinSimple).
Proximity ligation assay (PLA)
Protein‒protein interaction analysis was performed using the Duolink® PLA Kit (Sigma‒Aldrich; DUO96000 and DUO96040) strictly following the manufacturer’s operational protocol. The brain slices of the mice were pre-stained with TSA dye for pericytes (CD146, Abcam, ab75769), endothelial cells (CD31, Cell Signaling, 77699), and astrocytes (GFAP, Invitrogen, PA5-16291). After sodium azide neutralization pre-treatment, the antibodies, LRP1 (Abcam, ab92544), PACSIN2 (Invitrogen, PA5–99032), and Aβ (ABclonal, A24422), were conjugated to species-specific PLA probes (DUO96040). Subsequent procedures inclouding incubation, hybridization, ligation, amplification, and signal development—were conducted according to the kit’s standard protocol. Nuclei were counterstained with DAPI, and images were acquired via confocal microscopy.
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