“Drug‐Carrier” Synergy Therapy for Amyloid‐β Clearance and Inhibition of Tau Phosphorylation via Biomimetic Lipid Nanocomposite Assembly

Abstract Amyloid‐β (Aβ) toxicity is considered to be companioned by Tau phosphorylation in Alzheimer's disease (AD). The clinical AD therapy is usually subjected to low blood‐brain barrier (BBB) penetration and complex interaction mechanisms between Aβ and phosphorylated Tau. A “Drug‐Carrier” synergy therapy is herein designed to simultaneously target Aβ and Tau‐associated pathways for AD treatment. To imitate natural nanoparticle configuration, the endogenous apolipoprotein A‐I and its mimicking peptide 4F fused angiopep‐2 (Ang) are sequentially grafted onto lipid nanocomposite (APLN), providing liberty of BBB crossing and microglia targeted Aβ clearance. For synergy treatment, methylene blue (MB) is further assembled into APLN (APLN/MB) for Tau aggregation inhibition. After intravenous administration, the optimized density (5 wt%) of Ang ligands dramatically enhances APLN/MB intracerebral shuttling and accumulation, which is 2.15‐fold higher than that Ang absent‐modification. The site‐specific release of MB collaborates APLN to promote Aβ capture for microglia endocytosis clearance and reduce p‐Tau level by 25.31% in AD pathogenesis. In AD‐Aβ–Tau bearing mouse models, APLN/MB can relieve AD symptoms, rescue neuron viability and cognitive functions. Collectively, it is confirmed that “Drug‐Carrier” synergy therapy of APLN/MB is a promising approach in the development of AD treatments.

NFTs in the brain. [1] However, the 3xTg model costs enormous money and time because both Aβ and Tau pathology cannot be present in the brain until 9 months of age, [2] which limits the application in studies. According to previous reports, Aβ  (82 μmol L -1 ) was incubated in saline at 37 ℃ for 7 days to aggregate into Aβ 1-42 fibers. [3] To find out a feasible modeling dose of AD mice with two pathological markers (Aβ 1-42 aggregation and phosphorylated Tau), we referred to the Aβ 1-42 dose we reported before and set a series of OA concentrations of 1 μmol L -1 , 2 μmol L -1 and 4 μmol L -1 , [4] which named as Low, Middle and High modeling group, then mixed three concentrations of OA with Aβ 1-42 , respectively. According to the aggregation of Aβ 1-42 and phosphorylated Tau, the best modeling group was selected and used in subsequent experiments. During incubation at room temperature for 96 h, the changes of particle size andPDI were measured by DLS.

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The Open SPR instrument (Nicoya, Ltd., ON, Canada) was used to conduct surface plasmon resonance (SPR) analysis of the binding ability between nanocomposites and two forms of Aβ 1-42 aggregation.
The sensor chip was activated by injecting N-hydroxysuccinimide (NHS) and Ethyl (dimethylaminopropyl)-carbodiimide (EDC, 1/1, mol/mol), then 200 μL of Aβ 1-42 solution was introduced to reach the appropriate level of immobilization. The carboxyl group on the chip was blocked by injecting ethanolamine (pH 8.5). When the SPR instrument started, the flow rate of the running buffer which is PBS (0.01 mol L -1 , pH 7.0) was set at 20 μL min -1 .
Meanwhile, the regeneration buffer sodium dodecyl sulfate (SDS) solution (0.01 mol L -1 ) was running at 150 μL min -1 . Before the next injection, the sensor chip was equilibrated by PBS for 5 min. The binding ability between series concentrations of the nanocomposites and Aβ  were assessed and analyzed through Trace Drawer software.

SPR analysis of dynamic binding behavior of peptide and lipid vesicles
The LIP-1 sensor chip was equilibrated at room temperature, dock it into the apparatus and . [5] The uptake ratio (UR%) was calculated as follow: UR% = the GMFI of BV-2 cells / the GMFI of SH-SY5Y cells.

Colocalization assay
The BV-2 cells (2 × 10 5 cells well -1 ) were seeded on glass-bottom tissue culture dishes. The medium was replaced by an FBS-free medium with FAM-Aβ 1-42 (2 μg mL -1 ) in the presence of PLN/MB and APLN/MB before being cultured overnight. Lysosomes were stained with Lyso Tracker-Red for 45 min and nuclei were dyed by Hoechst33258 for 15 min, respectively.

OA-induced phosphorylated Tau of neuron cells
The SH-SY5Y cells at a density of 2 × 10 4 cells well -1 were cultured in 96-well plates for 24 h.

Flow cytometry analysis of cell apoptosis
The SH-SY5Y cells (1 × 10 5 cells well -1 ) were seeded onto 24-well plates and cultured for 12 h. Thereafter, cells were treated with OA (40 nmol L -1 ) to induce the phosphorylated Tau.
Meanwhile, cells were treated with free MB, PLN/MB and APLN/MB at MB concentration of 2 μg mL -1 for another 48 h, respectively. The SH-SY5Y cells without MB treatment were 9 used as control. Cells were resuspended and incubated with Annexin V-FITC/PI and propidium iodide for 10 min, followed by FACS analysis.
The photographs were taken and observed through an inverted fluorescence microscope.

Western blotting
The

Drug administration
The mice were randomly divided into six groups (n = 8

Morris Water Maze (MWM) and nest construction task
The Morris Water Maze test was employed to monitor the learning ability and spatial memory of mice. [6] The whole process consisted of a 5 day training and a trial day on the sixth day.
Mice would be put into a round pool, which was divided into 4 quadrants and remembered where the platform was by the symbol (square, circle, triangle and so on) on the wall during 5 training days. Before the training began, mice were transferred to the room for 2 h. The water temperature should be kept at (25 ± 1) ℃ and mice were dried after swimming. On the first 2 days, mice would be guided by a visible platform with a flag. Other 3 training days, the flag was removed and the platform was hidden under the water surface about 1 cm. Each mice was faced the wall of the pool to start swimming and the swimming track was recorded by a video-tracking system (DigBehv-MG, Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China). If the mice were failed to reach the platform within 1 min, they were guided to the platform and remained for 10 s. All the mice were trained for thrice, which started from a random place (Northeast, Northwest, Southeast and Southwest) of pool in a training day. On day 6, each mouse was allowed to swim for 1 min without the platform. The latency, swimming pathway and times of crossing in the target quadrant of mice were recorded and analyzed by an equipped software for the MWM.
The nest construction experiment could evaluate the cognitive ability of mice. A mouse was put into a cage with a corncob padding of about 1 cm thick. Before the experiment began, pieces of square towel (5 cm × 5 cm) were placed into the cage to assess nest-constructing behavior. [7] After 24 h, the nest was captured and scored as follows: 0 = the towel was still square and no obvious biting; 1 = the towel was torn into some big pieces but no gathering; 2 = the towel was torn into some big pieces and was concentrated in the cage; 3 = the towel was torn into some small pieces and was on one side of cage; 4 = the towel was torn into some 11 small pieces and was gathered in a corner. The scores were graded blindly by more than three experimenters.

Western blotting for tissues
When finished testing, the Sham and AD mice were sacrificed. Brain tissues were collected and stored at -80 ℃. The brain tissue homogenate was prepared as follow: the equal quality of RIPA containing PMSF, a protease inhibitor (1:100) and a phosphatase inhibitor (1:100) was added, then brain tissue was grinded by a tissue grinder (TL2010S, DHS Biological Technology Co., Ltd., Tianjin, China) at 1800 rpm for 5 rounds (work 60 s per round and 10 s interval). After centrifugation at 12500 rpm, 4 ℃ for 20 min, the supernatant was collected.
The protein in the supernatant was determined by BCA assay. The supernatant diluted with 4 × loading buffer was heated for 10 min and separated by 10% SDS-page. After electrophoresis, the protein was transferred to the PVDF membrane. Thereafter, the membrane was blocked by 5% BSA for 1 h at room temperature, incubated with the primary antibody as Anti-pTau Ser396 antibody ( For the bio-safety analysis, the mice were sacrificed and the major organs were collected for HE staining. Organs from the saline treated mice and AD mice were used as control.

Statistical Analysis
All the details about sample size, data presentation, statistical analysis and significant differences are provided in the figure captions. Unpaired two-tailed student's t-test was used for two-group comparison. One-way ANOVA, followed by Tukey post hoc analysis was used for multi-group comparison. The differences were displayed as significant for *p < 0.05, **p < 0.01 and ***p < 0.001. Data were expressed as mean ± standard deviation (SD). Statistical analysis of data was performed by Prism GraphPad 9 software.  The binding affinity of 4F peptide and αAng to lipid nanoparticles was measured by SPR, respectively. Figure S2. A) Representative images and B) scores of nest behavior for each group. Data were presented as mean ± SD (n = 8). *p < 0.05 and ***p < 0.001. Figure S3. Quantification results of p-Akt, p-GSK3β and p-Tau in mice brain. p-Akt, p-GSK3β and p-Tau were normalized to total Akt, GSK3β and Tau, respectively. Data were presented as mean ± SD (n = 3). *p < 0.05 and *** p < 0.001.