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Legumain-induced intracerebrally crosslinked vesicles for suppressing efflux transport of Alzheimer's disease multi-drug nanosystem

Brain barrier is both a protective permeability hurdle and a limitation site where therapeutic agents are excluded to enter the target region. Designing drug vehicle to overcome this notorious barrier bottle is challenging. Herein, we construct a stimuli-responsive self-assembled nanovesicle that delivers water-soluble drugs to prevent the efflux transport of brain barriers by responding to the endogenously occurring signals in Alzheimer's disease (AD) brain microenvironment. Once stimuli-responsive vesicles are accumulated in intracerebrally, the intrinsically occurring legumain endopeptidase cleaves the Ac-Ala-Ala-Asn-Cys-Asp (AK) short peptide on the drug vesicles to expose the 1,2 thiol amino group to cyclize with the cyano groups on 2-cyano-6-aminobenzothiazole (CABT) of the chaperone vesicles, thus triggering the formation of cross-linked micrometre-scale vesicles. Such a structural alteration completely prevents further brain barriers efflux. The superior neuroprotective capacity of cross-linked vesicles is validated in senescence accelerated mouse prone 8 (SAMP8). This smart multi-drug delivery vesicle is promising to serve as a powerful system for AD treatment and can be adapted for the therapy of other central nervous system (CNS) disorders.


Introduction
No clinical treatment drug for Alzheimer's disease (AD) could completely reverse the aging process over the past decade. During these barren years, drug hunters were largely focused on identifying agents that target either β-amyloid, tau protein, metabolic disorder or neurotransmitters [1,2]. Although biologists have been struggling to pinpoint what really causes the disease, the more urgent clinical management dilemma is how to deliver sufficient dose of therapeutic drugs into brain parenchyma to avoid the continued deterioration of the medium and advanced patients. However, the disappointing fact is that about 98% of small molecule drugs and virtually all large molecule drugs failed to enter the brain due to the brain barrier [3][4][5][6][7].
Polymer vesicles nanomedicine presents a promising opportunity to greatly boost AD therapy [8][9][10]. Indeed, intensive investigations have demonstrated that functionalized vesicles can be employed as promising delivery systems to enhance loading of hydrophilic and hydrophobic drug and thus protect the drug from enzymatic degradation in blood [11]. Brain barrier could protect neuron against neurotoxin, but this barrier bring large difficulty for drug brain parenchyma retention. Efflux pump distributed in the endothelial cell is emerging as a critical mechanism for nanomedicine brain efflux [12]. Higher brain endothelial cell internalization and exosmosis ratio of nanomedicine can be expected [13,14]. Yet, research results indicate that endothelial cell-penetrating properties of nanomedicine do not imply brain retention ability [15][16][17]. Targeting brain delivery does not solely increase penetration or suppress the efflux protein of BBB. The central mechanism by which modulate nanomedicine brain barrier transport and brain retention has been elucidated. Systems studies were performed to address the transport pathway, transport efficiency and thresholds for nanomedicine brain retention [18,19]. Indeed, nearly 75-95% of peak value nanomedicine that released in brain parenchyma was pushed out of the brain by efflux protein or paravascular glymphatic pathway [15,17,[20][21][22][23][24]. The multimode overlay of influx and efflux BBB transport leading to the nanomedicine brain retention ratio raised at first, then decreased, and the peak appeared at 1-2 h after nanomedicine taken [15,17,21]. Therefore, drug targets delivery will be the strategies those can manage nanomedicine to overcome efflux in brain parenchyma.
The different environment of the diseased cell that provide a unique opportunity for rational design of nanomedicine. Numerous studies have demonstrated that legumain has prominent role in AD pathogenesis, which has major clinical implications [25]. During ageing and other cellular stressors in the AD brain, legumain translocates from the endolysosome into the cytoplasmic space, and then legumain is widely distributed in brain parenchyma in AD patients but is not activated in CSF [26][27][28]. These understanding of the active leguamin and nanomendicne efflux mechanism that control brain retention, revealing the crucial role of reprogramming factors of legumain for target retention AD brain [29][30][31].
We hypothesize that legumain could be selected as a target to trigger in situ cross-linking of small size nanodrug particles to form large sized drug clusters and thus block the efflux of brain. Thus, three types of smart size switchable polymer vesicles loaded respectively with insulin, donepezil hydrochloride and memantine hydrochloride were designed and fabricated in this study. Among them, the insulin (INS) drug is used to down-regulate insulin resistance, and donepezil hydrochloride (DON HCl) is used to inhibit the breakdown of intracellular acetylcholine to maintain stable cholinergic content. Memantine hydrochloride (MEM HCl) is expected to reduce excitatory amino acid toxicity and prevent synaptic dysfunction caused by Ca 2+ influx [32][33][34][35][36]. The mixture of the V INS , V DON , V MEM vesicles and V CABT vesicles was named MEM/DON/INS ICV. When intrinsically legumain endopeptidase cleaves the Ac-Ala-Ala-Asn-Cys-Asp (AK) short peptide on surface of the (V INS , V DON , V MEM ) vesicles, then the exposure 1,2 thiol amino group could cyclize with the cyano group of 2-cyano-6-aminobenzothiazole (CABT) and thus triggering the formation of cross-linked vesicles, which could inhibit the brain efflux ( Fig. 1a and  b). These intelligent nanovesicles provide the possibility for synchronous delivery of different drugs that are precisely regulated to improve mitochondrial and synaptic functions from different mechanisms in neuronal cells. It is anticipated that this smart system could also enhance the brain retention of drugs, collectively improving the memory ability of AD patients.

Synthesis of the PCL-HEBIB macroinitiator
Briefly, 2-hydroxyethyl 2-bromoisobutyrate (HEBIB, 0.1 g, 0.47 mmol) and anhydrous ε-caprolactone (2.32 g, 20.2 mmol) were dissolved in refined toluene (6 mL) in a 25 mL flask and Sn(Oct) 2 (200 μL) were used as catalysts. The flask was then sealed and placed in an oil bath at 110 • C. The molar ratio of HEBIB/ε-CL was 1:45. After 12 h of polymerization, the reaction was terminated by exposure to air. The residue was dissolved in dichloromethane, precipitated from cold ethyl ether three times. After drying overnight in vacuum, the product was obtained with 85% yield and named PCL-HEBIB. A series of products were synthesized by varying the molar ratio of ε-CL/HEBIB.

Preparation of dextran-acryloyl chloride (Dextran-AC) macromonomer
Dextran (Mw = 2,000, 0.8 g, 4.9 mmol of anhydroglucose unit) was dissolved in N,N-dimethylformamide (DMF, 20 mL) and stirred at 0 • C, at the same time, triethylamine (Et 3 N, 50 μL) was added. Then, dichloromethane (8 mL) and acryloyl chloride (32 μL, 0.4 mmol) were mixed and slowly added dropwise to the flask. After 2 h, the flask was sealed at room temperature for 24 h. The mixture was thoroughly dialyzed against distilled water (MWCO: 1000 Da) for 72 h. After lyophilization, the product was obtained as a white solid with 77% yield and named Dextran-AC.

Synthesis of PCL-Dextran (PCL-Dex) copolymer
In a typical procedure，PCL-HEBIB (0.2 g), Dextran-AC (0.4 g) and CuBr (5.7 mg, 0.04 mmol) were dissolved in refined dimethyl sulfoxide (DMSO, 8 mL) in a 25 mL reaction tube. The mixture was repeated freezing and thawing three times. N, N, N ′ , N, 'N ′′ -pentamethyldiethylenetriamine (PMDETA, 6.9 mg, 0.04 mmol) was then dissolved in refined DMSO (0.5 mL) and added followed by freezing and thawing for another 3 times. The polymerization was carried out in an oil bath at 70 • C for 6 h. The resulting polymer was washed 5 times with ultrapure water to remove water-soluble compounds, and then dialyzed for 72 h (MWCO: 3500 Da). After lyophilization, the product was obtained as a white solid with 80% yield and named PCL-Dextran (PCL-Dex). Similarly, a series of PCL n -Dex were obtained by changing the molar ratio of PCL-HEBIB/Dextran-AC.

Polymers and nanoparticles characterization
The chemistry structures were examined by 1 H NMR (AVANCE 400 MHz, Bruker AXS Inc, Madison, Wisconsin). The hydrodynamic diameter was carried out with a Malvern Zetasizer nano ZS apparatus (Malvern Instruments, Malvern, USA). Nanoparticles were diluted with demineralized water, added onto copper grids and dried, then stained with uranyl acetate (0.2 wt%), and visualized using a Hitachi H-600 (Chiyoda, Tokyo, Japan) transmission electron microscope (TEM) operating at 200 kV.

Static light scattering (SLS) of PCL 45 -Dex 60 nanoparticles
Static light scattering (SLS) technique was employed to analyze the vesicular structure of the PCL 45 -Dex 60 polymer assemblies. The SLS data for PCL 45 -Dex 60 in water at 25.0 ± 0.1 • C was carried out and the intensity of scattered light at different angle was plotted against q 2 , where q is scattering vector magnitude (Guinier plot) [37,38].
Note: In SLS experiment, intensity was measured as a function of q (which was varied by angle θ). The variable q represents the scattering vector magnitude. It is given by the equation: Here n is the solution refractive index (1.332) and λ o is the incident wavelength, ie: 632.8 nm. The radius of gyration (R g ) was calculated by the equation: Here slope of the graph is R g 2 /3. In the case of PCL 45 -Dex 60 , Slope = 3340.4. So R g = 100.1 nm. 45 -Dex 60 -PGMA 12 -AK nanovesicles and PCL 45 -Dex 60 -PGMA 12

Preparation of drug encapsulated vesicles
Typically, donepezil hydrochloride (5 mg) and PCL45-Dex60-PGMA12-AK polymer (20 mg) were dissolved in DMSO (2 mL) and slowly added to 10 mL Milli-Q water at a rate of 10 μL s − 1 . The resulting solution was stirred for 8 h, dialyzed against Milli-Q water (MWCO: 3,500Da) for 48 h, named V DON and then lyophilized. The insulin-loaded vesicles (V INS ) and memantine hydrochloride-loaded vesicles (V MEM ) were prepared by the same operation. The freeze-dried V DON or V INS or V MEM (1 mg) was dissolved in DMSO (1 mL), respectively, diluted to 30 times, and the absorbance at 270, 276 and 365 nm was measured on a UV-1901 spectrophotometer (China).

Drug loading content
Similarly, RhB Vesicle AK was successfully prepared.

In vitro drug release studies
The V DON or V INS or V MEM vesicles (3 mL, 2 mg mL − 1 ) were taken in a dialysis bag (MWCO = 10,000 Da), respectively, and they were immersed in phosphate buffer saline (PBS, pH = 7.4, 97 mL) at 37 • C, then 10 units of enzyme were added and dialyzed with constant stirring [39,40]. At specific time intervals, 4.0 mL of the dialysate was withdrawn and replaced with an equal volume of fresh PBS buffer. The amount of drug released in each aliquot was measured by using absorption spectroscopy to quantify their percentage of cumulative release. The cumulative release rate of the drug in MEM/DON/INS ICV large nanoaggregates were detected with the same way.

Cell lines and animals
Human neuroblastoma cells (SH-SY5Y) and Red blood cell were purchased from Cell Bank of Chinese Academy of Sciences. Normal male aging mouse (Senescence accelerated-resistant mouse 1, SAMR1) and rapid male aging mouse (SAMP8) mice were purchased from Animal Breeding Center of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine.

Preparation of Aβ42 aggregates
Aβ 1-42 lyophilized powder and 300 μL 1, 1, 1, 3, 3, 3-hexafluoro-2propanol (HFIP) were injected into the reagent bottle and vortex. After mixing, it was allowed to stand at room temperature to obtain an Aβ-HFIP solution (1 mM). Prior to use, a small amount of DMSO was used for sonication, and PBS buffer was added and incubated for 1 week. The solution was diluted to the target concentration gradient with serum-free DMEM medium to used follow-up test.

Cell damage model
SH-SY5Y cells were maintained in DMEM medium, containing 10% fetal bovine serum (FBS) under the culture conditions of 5% CO 2 and 37 • C. Cells were harvested from flasks and plated in 96-well polystyrene plates with approximately 10 4 cells per well. Plates were incubated at 37 • C for 24 h to allow the cells to attach, followed by introduction of Aβ42 = 5 μM/well, 10 μM/well, 15 μM/well, 20 μM/well, 25 μM/well, respectively. After 6, 12, 24, 48 h, the cells were treated with 5 μL MTT solution (10 mg mL − 1 ) for 4 h and then 100 μL DMSO was added to each well. Plates were shaken at room temperature for 10 min to dissolve the crystals before the absorbance at 490 nm was measured using a Varioskan LUX (Thermo Scientific, USA). Averages of six replicate wells were used for each sample. drug is about 0.04 mg mL − 1 DMEM medium. After culture for 48 h at 37 • C, the MTT experiment was carried out in accordance with the above procedure.

Retention of RhB ICV and RhB Vesicle AK vesicles in SH-SY5Y cell by laser scanning confocal microscope (LSCM)
SH-SY5Y cells were transferred to laser confocal dish and incubated in DMEM. The induced cross-linked vesicles ( RhB ICV, the mixture of RhB Vesicle AK and V CABT vesicles) or noncross-linked Vesicle AK ( RhB Vesicle AK ) were added to the medium at a final concentration of 0.2 mg mL − 1 . After 2 h and 24 h incubation, SH-SY5Y cells were washed with PBS three times and fixed with 4% paraformaldehyde in PBS for 15 min at 37 • C, then stained with Hoechst 33258 (400 μL，10 μg mL − 1 ) to allow nuclear staining or LysoTracker Green (300 nM) to allow lysosome staining. Images of cells were taken with Nikon A1 Laser scanning confocal microscope (Japan).

Internalization of RhB ICV and RhB Vesicle AK nanovesicles by SH-SY5Y cells
SH-SY5Y cells were transferred to 6-well polystyrene plates with approximately 10 5 cells per well. RhB ICV or RhB Vesicle AK was added at the above concentrations.

Red blood cell aggregation
The red blood cells were transferred to 6-well polystyrene plates with approximately 10 5 cells per well, and then 0. . Mice of each group were administered nasally for 24 days at the dose of MEM was 0.033 mg drug/kg body weight/day, the dose of DON was 0.033 mg drug/kg body weight/day and the dose of INS was 0.9 IU/kg body weight/day. Then, the Morris water maze (MWM) was performed for mice learning and memory test.

Morris water maze (MWM)
The mice were tested in a water maze (1.3 m in diameter, temperature was maintained at 24 ± 1 • C). The platform (diameter of 15 cm) was fixed to beneath the water surface during the training period. Each subject was given 4 trials/day from different quadrants for 5 consecutive days with a 15-min intertrial interval. The maximum trial length was 60 s and if the mice could not find the platform within 60s, they were manually guided to catch the target platform. The trajectory of each mouse was recorded and analyzed for latency and swim speed. At 24 h after the last learning trial, a probe trial was presented during which time the platform was removed and the percentage of time spent in the quadrant, which previously contained the escape platform during task acquisition was measured over 60 s [25].

Western blot (WB)
Animals were euthanized, the hippocampus tissue of the SAMP8 mice was obtained. This tissue was thoroughly ground and centrifuged in the lysis buffer. An equal amount of protein sample (30 μg/sample) was processed at 97 • C for 6 min, centrifuge and transfer to film at room temperature. The poly (vinylidene fluoride) (PVDF) membrane was placed in a blocking solution (TBST/5% skim milk powder) and blocked at room temperature for 30 min. Primary antibody that diluted with blocking solution was added and incubated for 60 min at room temperature. After the 1×TBST washed, an appropriately diluted secondary antibody was added and incubated for 60 min. The development operation was carried out after rinsed 3 times with 1×TBST. The analysis was performed using Quantity one software, and the ratio of the control well (first well)/tubulin was taken as 1.

Malondialdehyde (MDA) content
According to the above method, the hippocampus was weighed, and 9 times volumes of physiological saline were added. The tissue was mechanically homogenized in an ice water bath condition, centrifuged at 2500 r minutes − 1 for 10 min, and the supernatant was taken for testing. After the addition of the MDA detection reagent, it was vortexed and placed in a boiling water bath for 30 min. The absorbance values of the supernatants at 532 nm were measured under UV-1901 spectrophotometer (China).

Acetylcholinesterase (AchE) activity
The tissue was treated in the same way, after AchE kit was added, and reacted accurately at 37 • C for 6 min. The absorbance values of the supernatants at 412 nm were measured under UV-1901 spectrophotometer (China).

Immunofluorescence
Animals were euthanized, the whole brain was harvested and fixed in 4% paraformaldehyde (PFA), then cut into hippocampus slices. Briefly, hippocampus slices were incubated for 2 h in a 1:1000 dilution of PSD-95 rabbit monoclonal antibody and reacted for 2 h in Akt mouse monoclonal antibody solution (1:1000 dilution), then immersed in fluorescence secondary antibody solution (1:500 dilution) for 1 h. Cell nuclei were labeled using 4 ′ ,6-diamidino-2-phenylindole (DAPI) for 10 min. These sections were observed under Nikon A1 Laser scanning confocal microscope (Japan).

NeuN staining and nissl staining
According to the above method, the thin hippocampus slice was incubated with anti-NeuN antibody diluted appropriately for staining 12 h at 4 • C. After washing, the slice was treated with secondary antibody for 35 min at room temperature and then exposed to 3,3 ′ -diaminobenzidine (DAB) to produce the chromogenic reaction. Similarly, nissl stained sections were prepared. The stained hippocampus slice was observed by Olympus CX41 microscope (Japan), and the NeuN positive cell numbers in the hippocampus were analyzed using ImageJ.

Hematoxylin eosin (HE) staining
Similarly, after the mice were sacrificed, the heart, liver, spleen, lungs, and kidneys were fixed and sliced. The sections were deparaffinized, washed in distilled water and incubated for 5 min in hematoxylin solution. Excess hematoxylin solution was washed away with running tap water and hydrochloric acid ethanol. The sections were then counterstained in an eosin solution for 3-4 min, washed in running tap water, dehydrated by graded alcohol, and fixed with a neutral resin. The pathological changes of the tissue were observed at 100X under an Olympus CX41 microscope (Japan).

Brain retention
The induced cross-linked vesicles ( RhB ICV) or noncross-linked Vesicle AK ( RhB Vesicle AK ) (40 μL) were injected into the SAMP8 mice by nasal administration, and ex vivo tissue imaging was performed at the indicated of 2 h and 12 h by KODAK In-Vivo Imaging System FX Pro (USA).

Hemolysis ratio
The 10 mL tubes were divided 8 groups: followed by introduction of 2.5 mL distilled water (positive controls), 2.5 mL saline (negative controls), 2.4 mL saline + 0. , respectively. The diluted blood (anticoagulant blood with 2% potassium oxalate 4:5 normal saline) was successively added into the solution of all groups. After incubation at 37 • C for 60 min, the blended liquid was centrifuged at 1000 rpm for 5 min. The supernatants were transferred into the 96-well plates, and the optical density (OD) values were measured at 545 nm with a microplate reader.

Determination of blood biochemical indicators
After the MWM experiment, the blood was collected from the orbital venous plexus of mice, and the serum was separated by centrifugation.

Statistical analysis
Statistical analyses for in vitro and in vivo experiments were performed by using Origin. The significance level was set at *P < 0.05, **P < 0.01, ***P < 0.005. Error bars represent the standard deviation of the mean (±SD), and P values were indicated in the figure captions and main text.
A series of PCL n -Dex 60 polymers were synthesized ( Fig. S1 and Table S1). We found that PCL 30 -Dex 60 formed clear solution, implying that hydrophilic dextran was predominant in the copolymer. While PCL 60 -Dex 60 solution showed poor stability as a small amount of sediment was observed after standing for 14 days. Further increasing PCL length, noticeable precipitation occurred to PCL 90 -Dex 60 solution after preparation immediately. The cause lied in that the excessively long hydrophobic chain segment of PCL reduced the stability of nanoparticles. Among them, only PCL 45 -Dex 60 formed stable suspension and no precipitation occurred even after 30 days of preparation. In order to verify the inner structure of PCL 45 -Dex 60 assemblies from a theoretical perspective, the radius of gyration (R g ) and hydrodynamic radius (R h ) were determined. The R g /R h ratios can provide the information on the solution assemblies of the polymer: R g /R h = 0.77 for hard spheres, R g / R h = 1 for vesicles, and R g /R h = 1.7 for coil-like structures [37,38]. The R h of the PCL 45 -Dex 60 assemblies was 99.1 nm (Fig. 3a, the half of hydrodynamic diameter value is R h ). The Rg was calculated from Guinier plot (Fig. 3b, the calculation for R g was detailed in the experimental section). It is noteworthy that the R g /R h value for the PCL 45 -Dex 60 polymer assemblies was ~1.0, suggesting the formation of vesicles. Fig. 3c-i showed the size and morphology of legumain-induced cross-linked nanoparticles as measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Before addition of legumain, the hydrodynamic diameter of the nanovesicles was in the range of 198-203 nm ( Fig. 3c and Table S2). With increasing the molar ratios of [Vesicle AK ]/[Vesicle CABT ] from 1:1 to 7:1, the size of the formed vesicles increased from 800 nm to more than 4.5 μm (Fig. 3d and Table S3). While the legumain-induced cross-linked Vesicle PCL45-Dex60-PGMA12-AK (Vesicle AK ) and Vesicle PCL45-Dex60-PGMA12-CABT (Vesicle CABT ) exhibited core-shell and spherical vesicles structure (Fig. 3e-i). Interestingly, after crosslinking, rod-like and vortex-like large vesicles appeared. When the molar ratio increased to 5:1, the large nanovesicles further fused and rearranged to form a network structure. This similar phenomenon was found in the self-assembly process of block copolymers, as the ratio of the hydrophobic/hydrophilic segments changed, the morphology of the formed nanoparticles changed from well dispersed nanoparticles to large compound vesicles (LCVs) [46][47][48][49]. The cross-linking should occur in brain parenchymal, large nanoaggregates act as reservoir for drug release. Under conditions that mimic the intracellular environment, the cumulative release rate of DON HCl from V DON was 89 ± 3.2% after 48 h. MEM HCl and INS released from the nanomedicine showed a similar kinetic profile (Fig. 4a, S2 and S13 and Table S4).

Neuroprotective effect and intracellular retention of induced crosslinked vesicles (ICV)
We further proceeded to examine the neuroprotective capacity and efflux inhibition effects of the functionalized vesicles on the SH-SY5Y cell culture. Hence, the protective effect of drug-loaded vesicles on Aβ42-induced SH-SY5Y cell injury was evaluated by cell viability via methylthiazolyl tetrazolium (MTT) assay (Fig. 4b), intracellular retention via flow cytometry (Fig. 4c) and laser confocal microscopy (Fig. 4d). Previous studies demonstrated that Aβ42-injured SH-SY5Y cell can mimic subcellular organ damage and stimulate legumain overexpression [22,23,50]. The incubation time and concentration of Aβ42 was determined by MTT test (Fig. S3). In this test, MV was the mixture of the V INS , V DON and V MEM vesicles. ICV was the mixture of the V INS , V DON , V MEM vesicles and V CABT vesicles (the molar ratio of V AK vesicles and V CABT vesicles is 5:1), those could be induced cross-linking by over-activated legumain. The cell viability of Aβ42-injured SH-SY5Y cells showed similar results over this time frame after MEM/DON/INS MV and drug co-encapsulation vesicle (MEM/DON/INS V) treatments. In contrast, the cell viability of Aβ42-injured SH-SY5Y cells treated with the MEM/-DON/INS ICV was very close to that of normal SH-SY5Y cells (Fig. 4b).
Thus, we have reason to believe that MEM/DON/INS ICV treatment can effectively repair SH-SY5Y cell damage caused by Aβ42.
In order to verify whether the best therapeutic effect of the induced cross-linked vesicles are related to the high retention rate of nanovesicles, the model drug rhodamine B (RhB) was selected to compare the differences between induced cross-linked vesicles ( RhB ICV, the mixture of RhB Vesicle AK and V CABT vesicles) and noncross-linked vesicle ( RhB Vesicle AK ). The SH-SY5Y cells were treated with RhB Vesicle AK or RhB ICV in DMEM for 2 h, 8 h and 24 h, and intracellular fluorescence intensity was quantitatively analyzed by flow cytometry. It was shown that the intracellular fluorescence intensity increased first and then decreased with time, and reached a maximum value at 8 h. Interestingly, the intracellular fluorescence intensity of SH-SY5Y cells treated with RhB ICV was 7-fold higher (p < 0.005) than that of RhB Vesicle AK treatment at 24 h ( Fig. 4c and S4).
To further determine if the cellular uptake of the nanovesicles experienced endosome/lysosome pathway, colocalization was performed by encapsulation of RhB for lysosomal staining. Fig. 4d showed that the red fluorescence was distributed in the SH-SY5Y cells at 2 h, and then no fluorescence was observed at 24 h, suggesting the RhB Vesicle AK could effectively cross the cell membrane and transported into endosome/lysosome, while the noncross-linked vesicle was difficult to be retained due to active efflux. By contrast, the RhB ICV also underwent endosome/lysosome pathway, with red fluorescence being detected in the SH-SY5Y cells internal area at 24 h, demonstrating the superior retention capacity of these vesicles. These results provided clear evidence that the legumain-induced cross-linked vesicles (ICV) had a better nerve cell retaining efficiency.

Improvement of memory ability of SAMP8 mice
We determined the improved memory function of senescence accelerated mouse prone 8 (SAMP8) after legumain-induced intelligent cross-linked nanovesicles treatment. The drug clinical standard dose was determined based on the treatment efficacy and its side effects, so each drug has its appropriate dosage. While, in large amount of the preclinical medicine literatures, the dose of medicine with different chemical design for improving the transporting and physiological barriers crossing capacity was designed to be same equivalent or approximate equivalent in the animal experiment [51][52][53]. And after the approximate equivalent drug administration in animal model, that the optimal medicine design system is summarized, evaluated and discussed. Furthermore, in the drugs combination treatment therapy that "cocktail" of drugs to achieve maximum benefit, how to observe the treatment efficacy, the possible treatment mechanism and the side effects of multiple drugs is a central theme. So, the combination treatment outcomes of multiple drugs were survey and analyze after the approximate equivalent drugs were administrated [54][55][56][57].
The memory ability of SAMP8 mice treated with drug-loaded vesicles was confirmed by Morris water maze (MWM) experiment after 24 days treatment. The memory ability of SAMP8 mice treated with saline or naked drugs was not improved, and the animals unable to find platform within the specified time (60 s). The memory ability of SAMP8 mice achieved a certain degree of recovery via MEM/DON/INS MV and MEM/DON/INS V treatments, and their escape latency was about 50 s ( Fig. 5a and c, S5 and S14-16). Comparatively, the mice with MEM/ DON/INS ICV treatment were able to travel shorter distances to reach the platform location, showing shortest escape latencies (~14 s, close to senescence-accelerated mouse resistant 1 (SAMR1) mice)relative to other treatment mice. Likewise, only MEM/DON/INS ICV treatment mice could accurately remember the platform location and exhibited a persistent memory for the target quadrant during a probe trial (Fig. 5b). In addition, similar swimming speeds (~10 cm/s, Fig. S6) indicated that differences in memory abilities were not caused by vision or motor dysfunction. Since the memory of mice changed over time with treatment, biomarker staining in hippocampus, like active nissl substance and nuclear protein (NeuN) were detected. The main function of the nissl substance is to synthesize the substances required for organelle renewal, which serve as markers of neuronal functional status [58]. The severe cell damage, large cell debris and less nissl substance were revealed in hippocampus slices by immunohistochemical analysis from SAMP8 mice treated with saline and naked drug (Fig. 5d). The nerve cells in the hippocampus arranged regularly, and the cytoplasmic nissl content increased after MEM/DON/INS MV treatment. More  importantly, a complete contour of hippocampus CA1 region and tightly arranged neurons could be observed, and no obvious cell damage or cell debris, a large amount of active nissl substance contained in cytoplasm after treating with MEM/DON/INS ICV. NeuN, a neuronal specific nuclear protein in vertebrates, was used to detect the number and distribution of neurons. The number of NeuN positive cells in SAMP8 mice hippocampus after MEM/DON/INS ICV treatment was 1642 ± 129, approaching to those of SAMR1 group (1663 ± 88). This value was 2 times higher than that of MEM/DON/INS MV group and 8 times higher than that of saline group (Fig. 5e and S7). The results indicated that neuronal damage in the brain of SAMP8 mice could be efficiently repaired via MEM/DON/INS ICV treatment.

Improvement of mitochondrial function and recovery of synaptic plasticity by induced cross-linked vesicles
In order to study the contribution of combination therapy to subcellular organelle repair from the perspective of signaling pathways, some of the mitochondrial and synaptic function-related biomarkers were examined, including protein kinase B (Akt), reactive oxygen species (ROS), postsynaptic density-95 (PSD-95), and acetyl cholinesterase (AchE). Among them, Akt protein was an important biomarker in PI3K/ Akt signaling pathway, and this pathway played a crucial role in glucose metabolism. The activation Akt protein was stimulated by insulin drug, which could promote the transition of glucose transporter (GLUT) to the cell membrane, enhance the uptake and utilization of glucose in the cell, maintain the stability of the energy metabolism cycle, facilitate the normalization of mitochondrial function, and reduce ROS content [34,35]. The Akt protein content of MEM/DON/INS ICV treatment mice was 29% higher than that of saline or naked drug treatment mice ( Fig. 6a and  b). While the ROS content from MEM/DON/INS ICV treatment mice was close to that of the SAMR1 mice (Fig. 6c). Previous studies suggested that AchE and PSD-95 protein played a key role in synaptic function, and AchE disrupted the stability of intersynaptic signaling by hydrolyzing acetylcholine to choline and acetic acid. AchE inhibitor-donepezil hydrochloride could effectively reduce the activity of acetylcholinesterase and maintain the stability of synaptic signals [33]. PSD-95 protein was a postsynaptic membrane compact that mainly formed a signal complex to regulate synaptic plasticity by binding different receptors. Post-synaptic membrane compact protectant-memantine hydrochloride (MEM HCl) could effectively attenuate the excitatory amino acid toxicity, and prevented post-synaptic membrane damage caused by Ca 2+ influx [32]. In comparison with the other treatment groups, the MEM/DON/INS ICV treatment group showed the lowest AchE activity (Fig. 6d) and the highest PSD-95 content ( Fig. 6e and f) in the hippocampus of SAMP8 mice. These values were close to those of SAMR1 mice, suggesting the pivotal role of MEM/DON/INS ICV therapy in repairing synaptic function damage. The immunofluorescence showed approximate results. The Akt protein (green fluorescence) and PSD-95 protein (red fluorescence) fluorescence signal intensity was enhanced in the hippocampus of MEM/DON/INS ICV treatment SAMP8 mice, in which the green fluorescence signal was overlapped with the blue fluorescence (nuclear) signal, suggesting widely distributed of Akt protein in cells (Fig. 6g). Based on the above analyses, it is reasonable to consider that the PI3K/Akt signaling pathway, cholinergic signaling pathway and excitatory amino acid signaling pathway play a vital role in the combination therapy of AD.

Brain retention
To further investigate the link between improvement of memory ability and brain retention of the intelligent drug-loaded vesicles, we used LC-MS and ex vivo tissue imaging to study the intracerebral retention. Previous studies demonstrated that the brain barrier influx and efflux of nanoparticles occurred simultaneously after 1-2 h [15,17,21]. With the passage of time, the efflux effect gradually dominated, and the brain retention rate of nanoparticles reached the lowest value after 6 h. The biodistribution of RhB Vesicle AK and RhB ICV vesicles at 2 h and 12 h share a common feature, that is, the brain and liver total retention constituted more than 91%, and the heart, spleen, lung, kidney and the blood retention make up was less than 9% (Fig. 7a-c). The data indicated that the nanovesicle brain accumulated efficiency depend on the combined effect of influx and efflux behavior of brain. The gulf between the well-designed legumain responsive RhB ICV vesicle and RhB Vesicle AK control nanovesicles was immense. The biodistribution of RhB Vesicle AK fluorescent nanovesicles in the brain was 39.0 ± 1.0%, as similar as SAMR1 group, after nasal administration 2 h, while the percentage of the RhB ICV fluorescent nanovesicles in the brain was 55.5 ± 1.4%. Yet the awareness of the tremendous differences between RhB Vesicle AK and RhB ICV in the administration 12 h biodistribution data, the fluorescence signal of RhB ICV group was 45.8 ± 0.9%, which was ~6.84-fold higher than that of RhB Vesicle AK group. As a supplement evidence, the quantitative analysis by HPLC-MS shown that the DON target brain retention efficiency of the MEM/DON/INS ICV group was about 26.38 ± 0.36% ID/mg at 12 h, which was about 17.0-fold higher than that of the MEM/DON/INS MV group (Fig. S8 and S9). Additionally, hemolysis assays demonstrated no hemolysis for MEM/DON/INS MV or MEM/-DON/INS ICV and the hemolysis ratio was lower than 1% (Fig. S10). Cerebral thrombosis or red blood cell aggregation did not occur (Fig. S11). MEM/DON/INS MV or MEM/DON/INS ICV did not have any toxic effect on liver and kidney functions and had no obvious toxicity to heart, spleen and lung of animals ( Fig. 7d and S12).

Conclusion
We have now demonstrated that high drug-retaining smart pathological signal-triggered cross-linked vesicles (MEM/DON/INS ICV) with both synaptic and mitochondrial repair functions are necessary and sufficient to improve memory capacity of the SAMP8 mice. Mechanistically, these novel nanovesicles can enhance brain retention of INS, MEM HCl and DON HCl, acting on the PI3K/Akt pathway, the excitatory amino acid pathway and the cholinergic pathway, respectively. More importantly, these smart nanovesicles promote the balance of energy metabolism, and prevent synaptic damage, which in turn protects neuronal cells and ensures the unobstructed transmission of signals between neurons. These findings provide not only a basis for the combined treatment of AD, but also the possibility of high intracerebral retention of the drug for other disease, indicating the potential importance of this intelligent drug delivery system.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.