Biomimetic ApoE-Reconstituted High Density Lipoprotein Nanocarrier

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Biomimetic ApoE-Reconstituted High Density Lipoprotein Nanocarrier for Blood-Brain Barrier Penetration and Amyloid Beta-Targeting Drug Delivery Qingxiang Song, Huahua Song, Jianrong Xu, Jialin Huang, Meng Hu, Xiao Gu, Juan Chen, Gang Zheng, Hongzhuan Chen, and Xiaoling Gao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00781 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Biomimetic

ApoE-Reconstituted

High

Density

Lipoprotein

Nanocarrier for Blood-Brain Barrier Penetration and Amyloid Beta-Targeting Drug Delivery ⊥

†⊥

†

†

†

†

† Qingxiang Song , , Huahua Song , , Jianrong Xu , Jialin Huang , Meng Hu , Xiao Gu ,

Juan Chen‡, Gang Zheng‡, Hongzhuan Chen†,*, Xiaoling Gao†,* †

Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong

University School of Medicine, 280 South Chongqing Road, Shanghai 200025, PR China ‡

Princess Margaret Cancer Centre, University Health Network, Canada

*

Corresponding author, [email protected]; [email protected]

⊥

Authors contributed equally

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Amyloid beta (Aβ) and its aggregation forms in the brain have been suggested as key targets for the therapy of Alzheimer’s disease (AD). Therefore, the development of nanocarriers which possess both blood-brain barrier permeability and Aβ-targeting ability is of great importance for the intervention of AD. Here we constructed a biomimetic nanocarrier named apolipoprotein E (ApoE)-reconstituted high density lipoprotein nanocarrier (ANC) from recombinant ApoE and synthetic lipids to achieve the above goals. α-mangostin (α-M), a polyphenolic agent which can inhibit the formation of Aβ oligomers and fibrils and accelerate Aβ cellular degradation, was used as the model drug. Compared with the control liposome, ANC demonstrated about 54-fold higher cellular uptake in brain endothelia cell line in vitro in an ApoE-dependent manner, and much higher brain delivery efficiency in vivo. Confocal microscopy analysis witnessed the penetration of ANC across the brain vessels and its accumulation at the surrounding of Aβ aggregates. Following the loading of α-M, the Aβ-binding affinity of the nanoformulation (ANC-α-M) was not reduced but even enhanced. The effect of ANC-α-M on facilitating the microglia-mediated uptake and degradation of Aβ1-42 was enhanced by 336% and 29-fold when compared with that of the non-treated control, and also much higher than that of ANC. Following intravenous administration for two to four weeks, ANC-α-M exhibited the most efficient efficacy in decreasing amyloid deposition, attenuating microgliosis and rescuing memory defect in SAMP8 mice, an AD mouse model. Taken together, the findings of this work provided strong evidences that the ApoE-based biomimetic nanocarrier could provide a promising platform for brain drug delivery towards the treatment of AD.

KEYWORDS: blood-brain barrier, amyloid beta, Alzheimer’s disease, nanocarrier, biomimetic, α-mangostin


Page 2 of 29

Page 3 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Today, more than 46 million people are living with dementia worldwide, and this number is estimated to increase to 131.5 million by 2050,1 among which Alzheimer’s disease (AD) is the most common form. The key pathological changes in AD are the cerebral accumulation of amyloid plaques, neurofibrillary tangles, glial proliferation and neuronal loss.2 Increasing evidences suggested that the initiating event in AD is related to abnormal processing of amyloid beta (Aβ) peptide, ultimately leading to formation of Aβ plaques in the brain. The toxicity associated to soluble Aβ oligomers formed on the pathway to the mature amyloid fibrils is especially highlighted.3,4 Therefore, Aβ and its aggregation forms have been suggested as key targets for AD therapy. The development of nanotechnology has provided useful strategies for Aβ-targeting

therapy.

Nanocarriers

such

as

anti-Aβ1-42

MAb-decorated

nanoliposomes,5 curcumin-decorated nanoliposomes,6 curcumin-conjugated magnetic nanoparticles,7 and phosphatidic acid and cardiolipin-based lipid nanocarrier

8,9

have

been developed and showed high affinity to Aβ and their aggregates. However, most of these nanocarriers lack permeability across the blood-brain barrier (BBB), which locates at the level of the cerebral microvasculature, is critical for maintaining the homeostasis of the central nervous system (CNS), but represents a major obstacle to the brain drug delivery. Therefore, the development of nanocarriers that possess both BBB permeability and high Aβ targetability is of great importance.10-12 Lipoproteins, natural nanoparticles, play a well-recognized biological role and are highly suitable as carriers for the delivery of therapeutic agents. By mimicking the endogenous shape and structure of lipoproteins, lipoprotein and lipoprotein-inspired nanoparticles can largely evade the mononuclear phagocyte system in the body's defenses. In particular, high-density lipoprotein (HDL), the smallest lipoprotein, is of special interest because of its ultra-small size and relatively long circulation half-lives. HDL has different exchangeable apolipoprotein components, ApoA-I and ApoE, which determine the structure and cholesterol transport abilities of HDL.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ApoA-I-based HDL majorly mediates peripheral cholesterol transport, whereas, ApoE-containing HDL plays a key role in cholesterol metabolism in the CNS.13 In addition to their primary function in lipid traffic throughout the brain.14 ApoE-HDL also plays a central role in Aβ metabolism.15 It has been shown that ApoE-HDL bound to Aβ and facilitate its clearance in a lipidation status (ApoE < ApoE-HDL) and ApoE isoforms (E4 < E3 < E2)-dependent manner.16 ApoE was also found to be co-localized with the newly formed Aβ deposits.17 Such information suggested that ApoE-HDL might be a nature nanocarrier for Aβ targeting. Besides, our previous work has witnessed the brain delivery of ApoE-conjugated albumin nanoparticles.18 Therefore, we hypothesized that in addition to the Aβ-binding affinity, ApoE-HDL might still possess BBB-penetrating activity, and might provide a novel nanoplatform for brain drug delivery towards the therapy of AD. However, the major limitation of utilizing native ApoE-HDL as a therapeutic nanoplatform originates from its source — cultured astrocytes or pooled fresh cerebrospinal fluid.19 This source is hard for scaling up, difficult for drug loading and is likely to produce quality variations among batches. Moreover, the application of native ApoE-HDL also poses the danger of transmitting infectious/pathogenic agents from the original biological systems.20 Therefore, ApoE-reconstituted HDL nanocarrier (ANC) prepared from commercially available recombinant ApoE and synthetic lipids, closely mimic the structure and metabolic behavior of its native counterpart, might represent a promising alternative to native ApoE-HDL.21 In our previous work, using ApoE3, the most predominant ApoE isoform found in healthy population, as the apolipoprotein component, we developed a novel nanomedicine that can bind to Aβ with high affinity and accelerate its clearance.11 In this work, in order to further test the hypothesis that ANC can be developed as a nanocarrier for BBB penetration and Aβ-targeting drug delivery, α-mangostin (α-M), a polyphenolic xanthone derivative from mangosteen, blocking the formation of both Aβ oligomers and fibrils, disturbing pre-formed fibrils and facilitating the cellular degradation of Aβ at low nanomolar concentrations,22,23 but exhibiting poor water solubility and low bioavailability,24 was used as the model drug. The Aβ-binding affinity and brain


Page 4 of 29

Page 5 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


delivery efficiency of ANC was evaluated both in vitro and in vivo. The effect of α-M-loaded ANC (ANC-α-M) on facilitating the microglia-mediated uptake and degradation of Aβ1-42, decreasing amyloid deposition, attenuating microgliosis and rescuing memory deficits in SAMP8 mice, an AD mouse model, was also demonstrated.

MATERIALS AND METHODS Materials. Dimyristoylphosphatidylcholine (DMPC) was provided by Avanti Polar Lipids Inc. (USA). ApoE3 was purchased from PEPROTECH Inc. (USA). Aβ1-42 peptides, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) were obtained from Invitrogen (Carlsbad, CA, USA). α-M (purity 98%) was purchased from Shanghai Liding Bio-technology co., LTD (Shanghai, China). Mouse monoclonal antibody 6E10 reactive to amino acid residues 1-16 of Aβ was obtained from Covance (Emeryville, CA, USA), Alexa Fluor 594-conjugated anti-mouse CD31 Antibody from Biolegend (San Diego, CA, USA) and anti-mouse CD45 antibody from BD Biosciences. Cell culture reagents were all provided by Thermo Fisher Scientific Inc. (product brand is Gbico, USA). Cell Lines. bEnd.3 cells were provided by Cell Institute of Chinese Academy of Sciences (Shanghai, China) and cultured in dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin at 37ºC in a humidified atmosphere containing 5% CO2. Animals. Balb/c nude mice (4-5 weeks, 20 ± 2 g, male) were obtained from Shanghai Laboratory Animal Center (Shanghai, China), and APP/PS1 transgene mice (9-month old) from Model Animal Research Center of Nanjing University. SAMP8 and SAMR1 mice (7-month old, 10-month old) were purchased from the Animal center of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. The animals were maintained in a special pathogen-free colony (IVC system, Tecniplast, Italy) at 25 ± 1ºC with free access to water and food. The protocol of animal experiments was approved by the Animal Experimentation Ethics



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Committee of Shanghai Jiao Tong University School of Medicine. Preparation and Characterization of ANC and ANC-α-M. ANC containing DMPC and ApoE3 was prepared as described previously.11 DiI, DiR-labeled ANC and ANC-α-M were prepared in a similar manner except that DiI (1%, w/w), DiR (1%, w/w) and α-M (12%, w/w) were included for the preparation of the lipid film. ANC-α-M was subjected to purification via a HiTrap column to remove the unentrapped α-M. Zetasizer Nano-ZS90 (Malvern Instruments, UK) was used to measure the zeta potential and size distribution of ANC. Transmission electron microscopy (TEM) and cryo scanning electronen microscopy (Cryo-EM) were used to characterize the morphology and structure of ANC. For TEM analysis, the samples were subjected to negative staining with 1.75% phosphotungstic acid and then observed via a Hitachi H-7650 transmission electron microscope (Hitachi, Inc., Japan). For Cryo-EM analysis, samples were prepared via a VITROBOT automated Cryo-EM sample preparation unit (FEI, Holland) and stored in liquid nitrogen until imaging. The images were acquired by FEI TECNAI G2 electron microscope (FEI, Holland) at 200 kV and −160ºC. To determine the encapsulation efficiency (EE) and the drug loading capacity (LC) of ANC-α-M, the nanoformulation was dissolved in acetonitrile and the concentration α-M was analyzed via the HPLC method as described previously.22 The DMPC concentration of the carrier was determined via LC-MS/MS analysis (Shimazhu 20AD-AB Sciex 4000 Mass Spectrometer) with a phenyl column with methanol: 0.1% formamide buffer (pH3.0) (98:2) as the mobile phase at the flow rate of 0.4 mL/min with an ESI source in positive ion mode. The curtain gas was set at 10 psi, ion source gas 1 at 50 psi, ion source gas 2 at 50 psi. The needle voltage was 4,500 V, gas temperature 600oC, collision gas 20 psi, declustering potential 170 V and collision energy 41 V. Multiple reaction monitoring (MRM) mode was applied for the detection of DMPC by using the transition of the m/z 678.5 to 184.1. EE was defined as the percentage of α-M being carried by the formulation and LC was defined as the weight ratio between the loaded α-M and the carrier. Cellular Uptake of ANC in bEnd.3 Cells. bEnd.3, a mouse brain endothelia cell


Page 6 of 29

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


line, was used to evaluate the interaction between ANC and brain endothelia cells. The cells were seeded at the density of 5 ×103 cells/well into 96-well plates, allowed to grow for 24 h, and then exposed to a series of concentrations of DiI-labeled ANC. For qualitative experiment, the ANC concentrations were ranged from 0.625 µg/mL to 20 µg/mL (DMPC concentration). After incubation for 3 h, the cells were washed twice with PBS, fixed in 4% formaldehyde for 15 min, stained with Hoechst 33258 for 15 min, and then subjected to ﬂuorescent microscopy analysis (LIFE EOVS® FL imaging system, USA). For quantitative analysis, the cells were incubated with DiI-labeled ANC (containing 5 µg/mL DMPC) at 37ºC and 4ºC for 10 min, 30 min, 1, 3 and 6 h, respectively. The cells were fixed with the nuclei stained as described above, then subjected to the analysis under a High Content Kinetic Scan (HCS) Reader (Thermo scientific, USA). To reveal the mechanism of cellular internalization of ANC in bEnd.3 cells, the cellular uptake of ANC was measured and compared in the absence or presence of various endocytosis inhibitors including 15 µM chlorpromazine, 10 µM colchicines, 10 µM filipin, 5 mM NaN3 + 25 mM deoxyglucose, 5 mM Amiloride, and 20 µg/mL ApoE3 protein. The cells were pre-incubated with the inhibitors for 1 h, respectively, then added with 5 µg/mL ANC, and further incubated for 3 h with the quantitative analysis performed as mentioned above. Brain Distribution of ANC Following Intravenous Administration. To determine the brain delivery efficiency of ANC, DiR was used as the fluorescent probe as the longer excitation and emission wave length (750/780 nm) of DiR would minimize the animal autofluorecence background and enable stronger tissue penetration, both of which are important for in vivo imaging. Twelve nude mice were randomly divided into two groups, and intravenously given with DiR-labeled ANC and ApoE3-free DMPC liposome, respectively. The fluorescent images were captured at 15 min, 1, 4, 10 and 20 h post injection with a the CRi Maestro® in vivo fluorescence imaging system (CRi, MA, USA). Four hours after administration, another three mice intravenously administrated with ANC or ApoE3-free DMPC



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

liposome were sacrificed with the brains harvested and imaged. Intracerebral Distribution of ANC in APPswe/PS1d9 (APP/PS1) Double Transgenic Mice. Nine-month old APP/PS1 mice (female) were intravenously administered with ANC (containing 4% DiI) at the dose of 10 mg/kg of DMPC. Two hours later, the mice were treated with the same protocol described previously with frozen brain slides prepared.25 To determine if ANC was delivered into the brain parenchyma and bound to Aβ aggregates in the brain, CD31 was used as the marker of blood capillaries, anti-Aβ antibody 6E10 was used for visualization of Aβ aggregates in the brain. The frozen slices were firstly rinsed three times with PBS, permeabilized with 0.2% Triton/PBS solution for 5 min, blocked with 20% goat serum for 1 h, and then incubated for 24 h at 4oC with anti-mouse CD31 antibody (1:100) or anti-amyloid beta antibody 6E10 (1:150). After rinsing three times with PBS, the slides were stained with Alexa Fluor® 488-conjugated Goat Anti-Mouse IgG secondary antibody (Invitrogen) for 1 h and then with DAPI for 8 min before the observation under a Zeiss LSM 710 microscope. Preparation of Aβ β1-42 Monomers and Oligomers. Aβ1-42 was firstly dissolved in hexafluoroisopropanol (HFIP) at 1 mg/mL. For the preparation of Aβ1-42 monomers, the solution was subjected to solvent evaporation, and the resulting peptide film was resuspended in DMSO to the final concentration of 5 mM with the solution further bath sonicated for 10 min. For the preparation of Aβ1-42 oligomers, the Aβ1-42 monomer solution (5 mM) was diluted to 100 µM with the pH 7.4 phosphate buffer containing 150 mM NaCl, and the solution was further incubated for 24 h at 4ºC. 11,26 SPR Analysis. For SPR analysis, Aβ1-42 monomers and oligomers were immobilized on a CM5 sensor chip (GE) using the amine coupling reaction as described previously with minor modification.26 Briefly, 0.2 M EDC and 0.05 M NHS were used for surface activation, Aβ1-42 monomers and oligomers (23 µM in acetate buffer pH 4.0) were injected for 420 s at the flow rate of 30 µL/min, and the remaining activated groups were blocked with ethanolamine, pH 8.5. The upstream parallel flow cell immobilized with BSA and then blocked with 1 M ethanolamine was used as the control surface. The binding was conducted in PBS (0.01 M, pH 7.4)


Page 8 of 29

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with the analytes (containing 0-100 nM ApoE3) injected into the flow system at the flow rate 30 µL/min. Dissociation was conducted in the same buffer for 6 min, and the chip was then regenerated with 2 M Glycine-HCl (pH 2.0). The calculation of the kinetic constants of binding was performed via BIA T200 evaluation software using the 1:1 Langmuir binding model. BV-2 Cells Uptake and Degrade of Aβ1-42 after Treated with ANC-α-M. BV-2 cells, a mouse cell line of microglia, were employed to assess how ANC-α-M affects microglia uptake and degradation of Aβ1-42. For the quantitative analysis of cellular uptake, BV-2 cells were seeded at the density of 1 ×104 cells/well into 96-well plates and allowed to grow for 24 h. After that, the cells were added with ANC-α-M and ANC (containing the same concentration of DMPC) at the α-M concentration 0, 25, 50, 100, and 200 ng/mL for 24 h, and then co-incubated with FAM-Aβ1-42 for 3 h. The fluorescent intensity of FAM-Aβ1-42 in the cell was quantified via the HCS analysis as described above. For the analysis of the cellular degradation of Aβ1-42, BV-2 cells were seeded into 6-well plates at the density of 2 ×105 cells/well and allowed to attach for 24 h. The BV-2 cells were then added with ANC or ANC-α-M at 0, 50, or 100 ng/mL for 24 h, and further co-incubated with Aβ1-42 (2 µg/mL) for 3 h. The concentration of Aβ1-42 remained in the media and in cell lysates was determined via an ELISA kit (Invitrogen Carlsbad, CA, USA). Treatment of AD Model Mice with ANC-α α-M. For immunohistochemical analysis, ten-month old male SAMP8 mice were daily and intravenously given with normal saline, α-M solution, ANC-α-M at the α-M dose of 0.5 and 2 mg/kg or ANC at the same DMPC dose with 0.5 mg/kg of ANC-α-M, for 2 weeks. For MWM test, seven-month old male SAMP8 were divided into four groups randomly and intravenously administered with saline, α-M solution (0.5 mg/kg/day), ANC (DMPC dose equal to 0.5 mg/kg ANC-α-M) or ANC-α-M (0.5 mg/kg/day) for 4 weeks. Age-matched SAMR1 mice administered with normal saline served as the normal control. Immunohistochemical Analysis. Following the drug treatment, the mice were anesthetized and heart perfused with cold saline. The brains were collected and fixed



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 µm. For immunohistochemistry, the slides were incubated in 70% citric acid for 3 min, and then in methanol containing 1% peroxide for 10 min. After microwaved in distilled water for 3 min, the slides were blocked in 20% normal goat serum solution containing 0.1% Triton X-100 for 1 h, and then incubated with primary antibody in the blocking solution overnight at 4oC. The 6E10 antibody against Aβ was used to stain Aβ plaques, and anti-CD45 antibody to stain the activated microglia. The antigens were detected by secondary antibodies using standard ABC-DAB methods, the images were captured via a microscope (Leica DM2500P with Leica DFC320 digital camera) and analyzed with Image Pro-Plus software (Media Cybernetics, Silver Spring, MD). MWM Test. MWM test was performed as described previously to evaluate the spatial learning and memory of AD model mice after the treatment.22 The mice were trained four times a day for five days. A computer-controlled tracking system (Shanghai Jiliang Software Technology Co., Ltd.) was used to record the escape latency and swimming route. On the sixth day, with the platform removed, the mice was placed into the tank from the same fixed positions and allowed to swim freely for 60 s with the times of platform crossing recorded. Statistical Analysis. The data were expressed as mean ± standard deviation (SD). Comparison between two groups was analyzed via two-tailed student t test, among multiple groups via one-way ANOVA followed by Bonferroni test, and statistical significance was defined as p < 0.05.

RESULTS Preparation and Characterization of ANC-α α-M. ANC containing DMPC and full length recombinant ApoE3 (5:1, w/w) were prepared as described previously.11,27 ANC-α-M was prepared by mixing α-M with DMPC for the formation of lipid membrane. Dynamic light scattering (DLS), TEM and Cryo-EM were used to characterize the size distribution, zeta potential and morphology of ANC and


Page 10 of 29

Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ANC-α-M. DLS analysis showed that the size of ANC was 27.40±9.70 nm with a relatively small size distribution (PDI=0.22), and the zeta potential of ANC was – 9.83±0.45 mV. Fluorescent labeling didn’t change either the size or the zeta potential of ANC. Following the loading of α-M, the size of ANC-α-M slightly increased (35.95±9.05 nm) while the zeta potential became more negative (–19.96±0.50 mV) (Table 1). Under TEM, ANC presented as stacking nanodiscs with a hydrodynamic long diameter of 10-20 nm. Such stacking was less seen in ANC-α-M (Figure 1C), which could be resulted from its more negative surface charge. Cryo-EM was used to further characterize the structure of ANC and ANC-α-M. As shown in Figure 1D, both strip and circular projections with a ring of high density were observed in both ANC and ANC-α-M, indicating the similarity of their structure. The drug loading capacity of ANC-α-M was 10.20±0.51% with a relatively high encapsulation efficiency, 82.60±5.30%.

Figure 1. Particle size, morphology and structure of ANC and ANC-α-M. (A) Size distribution of ANC measured by dynamic light scattering, (B) size distribution of ANC-α-M measured by dynamic light scattering, (C) morphology of ANC and ANC-α-M observed under transmission electron microscope after negative staining with phosphotungstic acid (1.75%, w/v), Bar, 60 nm, and (D) structure of ANC and ANC-α-M observed under cryo scanning electronen microscope. Bar, 50 nm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 29

Table 1. Particle Size and Zeta Potential of ANC and ANC-α-M Particle size (nm)

PDI

Zeta potential (mV)

ANC

27.42±9.70

0.22

–9.83±0.45

DiI-loaded ANC

30.64±7.53

0.28

–9.20±0.60

DiR-loaded ANC

28.60±4.46

0.33

–10.77±0.68

ANC-α-M

35.95±9.05

0.23

–19.96±0.50

DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiR, 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide. Cellular Uptake of ANC in bEnd.3 Cells. In order to evaluate the interaction between ANC and brain endothelial cells, bEnd.3, a mouse brain endothelia cell line confirmed by the observed expression of von Willebrand factor and uptake of fluorescently-labeled low density lipoprotein (LDL), was here used as the cell model. DiI, a long-chain dialkylcarbocyanine lipophilic tracer widely used for liposome and cell plasma membrane labeling that generally not transfer from the labeled to unlabeled ones, was utilized to label ANC at the amount of 1% to total lipid. Both qualitative and quantitative data showed that the cellular uptake of ANC was much higher than that of the ApoE3-free DMPC liposome (54-fold higher at the DMPC concentration 20 µg/mL after 3-h incubation) in a concentration, time and temperature-dependent manner (Figure 2). Mechanism analysis showed that the cellular internalization of ANC at 37oC was higher than that at 4oC, and inhibited by the presence of 2-deoxyglucose (DOG, 25 mM) and sodium azide (NaN3, 5 mM). Excess ApoE3 (20 µg/mL) also significantly inhibited the cellular uptake of ANC. Amiloride (5 mM) and chlorpromazine (15 µM) effectively reduced ANC uptake as shown in Figure 3 whereas colchicine (10 µM) did not inhibit the cellular association of ANC but reduced its internalization. In contrast, filipin (10 µM) did not affect the cellular uptake of ANC (Figure 3).


Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Cellular uptake of DiI-labeled ANC and ApoE3-free DMPC liposome, respectively. (A) Qualitative fluorescent images taken after incubation at 37oC for 3 h. Red, DiI-loaded ANC/ ApoE3-free DMPC liposome (containing DMPC 0.625, 5, 20 µg/mL); blue, nucleus. Bar, 200 µm. (B) High content scanning quantitative analysis of cellular uptake of DiI-labeled ANC and ApoE3-free DMPC liposome (containing DMPC 5 µg/mL), respectively, after incubation at 37oC for 3 h; (C) High content scanning quantitative analysis of cellular uptake of DiI-labeled ANC/ApoE3-free DMPC liposome (containing DMPC 5 µg/mL) after incubation at 37 and 4oC, respectively, for 10, 30 min, 1, 3 or 6 h. ***p < 0.001, significantly different with that of ApoE3-free DMPC liposome at the same temperature and same concentration.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Cellular uptake of DiI-labeled ANC in the presence of various endocytosis inhibitors. (A) Qualitative fluorescent images taken after incubation at 37oC for 3 h in the presence of various endocytosis inhibitors or at 4oC. Red, DiI-loaded ANC; blue, nucleus. Bar, 100 µm. (B) High content scanning quantitative analysis of cellular uptake of DiI-labeled ANC (containing DMPC 5 µg/mL) after pretreating with various endocytosis inhibitors at 37oC for 1 h, then co-incubation at 37oC for 3 h, ***p < 0.001, significantly different with that of non-inhibited control.

Brain Distribution of ANC after Intravenous Administration. To determine the brain delivery efficiency of ANC after intravenous administration, in vivo fluorescent imaging analysis was performed using near infrared fluorescent probe, DiR, also a long-chain dialkylcarbocyanine lipophilic tracker, to label ANC for minimizing the autofluorecence background. Following intravenous injection, the DiR signal in the brains of those animals treated with ANC at all the time points post-administration was higher than that of those treated with ApoE3-free DMPC liposome at the same DiR dose (Figure 4A). Four hours after dosing, three ANC-treated nude mice were euthanatized with the brains collected and imaged, and also obviously stronger


Page 14 of 29

Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fluorescent signal of the nanocarrier was found in the brains of these animals than that in those animals treated with ApoE3-free DMPC liposome (Figure 4B).

Figure 4. Brain distribution of ANC following intravenous administration. Twelve nude mice were divided into two groups randomly, and intravenously administrated with DiR-loaded ANC and ApoE3-free DMPC liposome via the tail vein, respectively. (A) In vivo distribution of DiR-loaded ANC and ApoE3-free DMPC liposome at 15 min, 1, 4, 10 and 20 h after intravenous administration in nude mice, n=3. (B) Fluorescent signal of DiR-loaded ANC and ApoE3-free DMPC liposome in the animal brain at 4 h after intravenous administration, n=3.

Intracerebral Distribution of ANC in APP/PS1 Mice. In order to justify if ANC can be delivered into the brain parenchyma following intravenous administration, vascular visualization was performed by combining immunofluorescence technique with confocal microscopy analysis using CD31 as the marker of brain blood capillaries. Most of the ANC was found to penetrate through the brain blood vessels and well dispersed in the parenchyma (Figure 5).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Penetration of ANC (red) across the brain vessels (green) following intravenous administration. Nine-month old APP/PS1 mice were intravenously administered with ANC (containing 4% DiI) at the dose of 10 mg/kg of DMPC. Two hours later, the brains of the mice were collected and sectioned. To study if ANC was delivered into the brain parenchyma, CD31 was used as the marker of brain blood vessels. Bar, 40 µm. The in vivo Aβ-targeting efficiency of ANC was evaluated using Aβ immunofluorescent analysis. It was found that following delivery into the brain parenchyma, in both the cortex and hippocampus, the major sites of Aβ aggregation, DiI-labeled ANC (red) was found highly accumulate at the surrounding of Aβ aggregates which was visualized by anti-Aβ antibody 6E10 immunofluorescent signals (green) (Figure 6).

Figure 6. In vivo accumulation of ANC (red) at the surrounding of Aβ aggregates (green) following intravenous administration (A) in the cortex and (B) in the hippocampus. Nine-month old APP/PS1 mice were intravenously administered with ANC (containing 4% DiI) at the dose of 10 mg/kg of DMPC. Two hours later, the brains of the mice were collected and sectioned. To study the Aβ-targeting efficiency of ANC, anti-Aβ antibody 6E10 was used for visualization of Aβ aggregates in the brain. Bar, 40 µm.


Page 16 of 29

Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ANC-α-M Bound to Aβ1-42 Monomers and Oligomers with High Affinity. The direct interactions between Aβ1-42 monomers, oligomers and ANC or ANC-α-M were characterized via surface plasmon resonance (SPR) analysis. Aβ1-42 and ANC or ANC-α-M interacted with each other in an ApoE-concentration-dependent binding manner. The binding affinity constant (KD) values of ANC to Aβ1-42 monomers and oligomers were 4.85±2.91 nM and 7.93±0.98 nM, respectively, while that of ANC-α-M to Aβ1-42 monomers and oligomer were 0.39±0.16 nM and 1.34±0.73 nM (Figure 7), respectively, suggesting that ANC-α-M displays higher Aβ binding affinity than the blank nanocarrier, ANC.

Figure 7. Surface plasmon resonance analysis of the ApoE-concentration-dependent binding of ANC and ANC-α-M to Aβ1-42 monomers and oligomers. (A) Binding of ANC to Aβ1-42 monomers. (B) Binding of ANC to Aβ1-42 oligomers. (C) Binding of ANC-α-M to Aβ1-42 monomers. (D) Binding of ANC-α-M to Aβ1-42 oligomers. The kinetic constants of binding were calculated via BIA evaluation software using the 1:1 Langmuir binding model, n=3.

ANC-α α-M Accelerated Microglia-Mediated Uptake and Degradation of Aβ1-42. To evaluate the effect of ANC-α-M in accelerating the cellular uptake and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

degradation of Aβ1-42, BV-2 cells were pre-incubated with various concentrations of ANC-α-M for 24 h and then co-incubated with FAM-Aβ1-42/Aβ1-42 for 3 h in the presence of the formulations. ANC containing the same amount of DMPC was applied as the control formulation. It was found that in the presence of ANC-α-M, the cellular uptake of FAM-Aβ1-42 increased in an α-M concentration-dependent manner. At the α-M concentration of 50 ng/mL and 200 ng/mL, the cellular uptake of FAM-Aβ1-42 increased by 160% and 336%, respectively, compared with that of the non-treated control. In contrast, ANC containing the same dose of DMPC only enhanced the cellular uptake of FAM-Aβ1-42 by 41% and 77%, respectively (Figure 8A). To evaluate the efficiency of ANC-α-M in facilitating the cellular degradation of Aβ, the level of Aβ1-42 in the culture media and cell lysates were determined via enzyme-linked immunosorbent assay (ELISA) (Figure 8B and Figure 8C), and the total Aβ degradation rate was calculated. In the absence of ANC and ANC-α-M, following 3-h incubation in BV-2 cells, the Aβ degradation rate was only 2%. But following the treatment with ANC-α-M at the α-M concentration of 50 ng/mL and 200 ng/mL, the Aβ degradation rate reached to 33% and 60% (increased by 15.5-fold and 29-fold, respectively), respectively, which was also significantly higher than that in the presence of blank ANC containing the same amount of DMPC (20% and 48%, respectively) (Figure 8D). In addition, the fate of ANC and ANC-α-M after Aβ degradation was also studied using 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO, green) as the fluorescent probe (Table S1, Supporting Information) and LysoTacker Red as the indicator of lysosome. It was found that despite the higher cellular uptake of ANC-α-M, both ANC and ANC-α-M were also transported to the lysosome for degradation after cellular internalization (Figure S1, Supporting Information).


Page 18 of 29

Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. ANC-α-M enhanced the cellular uptake and degradation of Aβ1-42 in BV-2 cells. The cells were treated with ANC-α-M or ANC (containing the same concentration of DMPC) at the α-M concentration of 0, 25, 50, 100 and 200 ng/mL for 24 h, then co-incubated with FAM-Aβ1-42 for 3 h. (A) The fluorescent intensity of cellular internalized FAM-Aβ1-42 in BV-2 cells, acquired via high content scanning analysis. (B) Concentration of Aβ1-42 remained in the culture media. (C) Concentration of Aβ1-42 in the cell lysates. (D) Total Aβ1-42 degradation rate in the presence of ANC or ANC-α-M. *p < 0.05, **p < 0.01, ***p < 0.001, significantly different with that of ANC at the same DMPC concentration.

ANC-α α-M Decreased Amyloid Deposition and Attenuated Microgliosis in SAMP8 Mice. To evaluate the efficiency of ANC-α-M in alleviating AD pathological process, senescence-accelerated mouse-prone 8 (SAMP8), which shows age-related cognitive decrease relevant to the changes of gene expression and protein abnormalities in AD,28 was applied as the animal model. Senescence-accelerated mouse resistant 1 (SAMR1) mice at the same age was used as the normal control. Anti-Aβ immunostaining analysis showed that compared with that in the brains of the normal saline (NS)-treated SAMP8 mice, the level of amyloid plaque in the brains of those animal following 2-week treatment of ANC-α-M (0.5 and 2 mg/kg, respectively)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was markedly decreased (72% and 91% of decrease, respectively) (Figure 9A). In addition, compared with that in the brains of the NS-treated ones, an 85% and 91% decrease in the CD45-positive activated microglia were also found in the brains of those ones administered with ANC-α-M at the dose of 0.5 and 2 mg/kg α-M, respectively (Figure 9B). In contrast, only 34% and 42% decrease in amyloid plaque load, and 25% and 32% decrease in microgliosis were found in the α-M solution-treated SAMP8 mice (0.5 and 2 mg/kg, respectively). Following the treatment with ANC, 64% decrease in amyloid plaque load and 84% decrease in microgliosis were observed.

Figure 9. ANC-α-M decreased amyloid deposition (A) and attenuated microgliosis (B) in the brains of SAMP8 mice. Ten-month old SAMP8 mice (n=3) received 2-week daily intravenous injection of α-M solution, ANC-α-M at the α-M dose of 0.5 and 2 mg/kg and ANC (DMPC dose equals to 0.5 mg/kg ANC-α-M), respectively. Age-matched SAMP8 and SAMR1 mice dosed with normal saline (NS) were used as the negative and normal control, respectively. The brain sections (4 µm) were immunostained with anti-Aβ antibody 6E10 and anti-CD45 antibody, respectively,


Page 20 of 29

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and quantitatively analyzed with Image Pro-Plus software. Bar, 40 µm. **p < 0.01, *** p < 0.001, significantly different with that in SAMP8 mice treated with NS, #p < 0.05, ###p < 0.001, significantly different with that in the SAMR1 mice treated with NS.

ANC-α-M Rescued Memory Defect in SAMP8 Mice. We then tested the effects of ANC-α-M on rescuing the memory defect in AD model mice. SAMP8 mice (7 month-old) showed spatial learning impairments in MWM task. The treatment with α-M solution and ANC slightly alleviated such defect in spatial learning and memory by shortening the escape latency and prolonging the time spent in a targeted quadrant after removing the platform. Following the administration with ANC-α-M (dose at 0.5 mg/mL α-M), the SAMP8 mice demonstrated significantly decreased escape latency at the fourth and the fifth day, and exhibited more active exploration and increased the number of platform crossings at the final day when the platform was removed (Figure 10).

Figure 10. ANC-α-M alleviated memory defect in SAMP8 mice. SAMP8 mice (7-month old) were intravenously given with ANC-α-M at the dose of 0.5 mg/kg α-M



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

daily for 4 weeks with age-matched SAMR1 given with normal saline (NS) as the normal control, and SAMP8 mice given with normal saline (NS), ANC (DMPC dose equals to 0.5 mg/kg ANC-α-M) and α-M solution at the α-M dose of 0.5 mg/kg as the negative control, nanocarrier control and free drug control, respectively. n=8. (A) Escape latency; (B) Times of platform crossing at the final day with the platform removed; (C) Representative swimming path. **p < 0.01, ***p < 0.001 statistically different with that of the SAMP8 mice treated with normal saline.

DISCUSSION AD is the most common form of dementia worldwide, representing the most challenging areas in modern medicine. Accumulating evidences indicate that Aβ accumulation in the CNS plays a key role in AD pathogenesis.29,30 Therefore, targeting brain Aβ and its aggregation forms have been actively pursued for therapeutic and/or diagnostic purposes.31 However, delivery of therapeutic/diagnostic agents into the CNS is limited by the existence of BBB. Advances in nanotechnology are now exerting a significant impact in neurology.10 and the development of properly-designed nanoformulations which allow both BBB crossing and Aβ targeting is expected to play an important role in AD therapy. In this study, we constructed a bio-inspired drug delivery system — ANC, which is expected to possess both Aβ-binding affinity and brain delivery efficiency, aiming at providing a useful nanocarrier for anti-AD drug delivery. ANC was here prepared by using commercially available synthetic lipids and recombinant apolipoprotein by firstly preparing liposome and then allowing auto-assembly between liposome and ApoE. As liposome has been widely used in clinical practice, the liposome production technology was well-established and well scale-up. ApoE automatically assembles to liposome due to its natural property, which could be also easy to achieve in the universal industrial condition. Such preparation procedure for ANC is flexible and also easy for scaling-up and reproducibility, which would endow ANC with more chances to reach the clinic. The obtained ANC exhibited the same discoidal structure as nascent HDL.32 Following the loading of α-M, a polyphenolic agent that blocking the formation of both Aβ oligomers and fibril, disturbing pre-formed fibrils and facilitating the cellular


Page 22 of 29

Page 23 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Aβ degradation, as demonstrated by TEM and Cryo-EM, the morphology of the nanostructure was generally maintained while its zeta potential became more negative, indicating that at least some α-M might be loaded by inserting into the lipid membrane of ANC-α-M. Interestingly, SPR analysis showed that α-M loading did not reduced but enhanced the Aβ-binding affinity of ANC (Figure 7). The following reasons could contribute to the higher binding affinity of ANC-α-M to Aβ. Firstly, following α-M loading, the zeta potential of ANC decrease from–9.83±0.45 mV to – 19.96±0.50 mV. The more negative surface charge probably contributes to the more efficient binding of ANC-α-M to Aβ monomer and oligomers. In addition, after loading α-M, ANC-α-M tend to disperse better than ANC (Figure 1C). The higher dispensability is also likely to raise the chance of ANC-α-M to bind to Aβ. And lastly, α-M itself possessed binding affinity to Aβ monomers and Aβ oligomers,23 their incorporation into the lipid membrane of ANC might also contribute to the enhanced Aβ-binding affinity of ANC-α-M. ANC facilitated the brain clearance of Aβ majorly via microglia-mediated Aβ uptake and degradation.11,26 α-M enhanced the cellular uptake and degradation of Aβ by up-regulating the expression of low-density lipoprotein receptor in microglia.22 To testify if the loading of α-M into ANC would accelerate the microglia-mediated Aβ uptake and degradation, in vitro analysis was performed using BV-2 cells as the cell model, finding that ANC-α-M facilitated the cellular uptake and degradation of Aβ1-42 by 336% and 29-fold, respectively, when compared with the non-treated control, and also much higher than that of ANC (Figure 8). Accessing the brain is one of the major prerequisites for ANC to mediate Aβ-targeting drug delivery. The expression of receptors to ApoE — low density lipoprotein receptor and low density lipoprotein receptor-related protein 1 were extensively observed on BBB. Receptor-mediated transcytosis of ApoE-modified albumin nanoparticles and low density lipoprotein across the BBB had also been reported.18 ANC, lipidated ApoE, which possesses higher affinity to these receptors than ApoE itself and smaller particle size than the above-mentioned nanostructures, is expected to exhibit higher BBB permeability. To justify this hypothesis, the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 29

interaction between ANC and the brain capillary endothelial cells — immortalized murine brain endothelial cell line bEnd.3 was studied here. The cellular uptake of ANC was found to be in an ApoE concentration, time and temperature-dependent manner, suggesting that this was an active transport process mediated by ApoE. In addition, in vivo fluorescent imaging analysis found that following intravenous injection, the fluorescent signal of DiR in the brains of those animals treated with ANC at all the time points post-administration was much higher than that in those animals treated with ApoE3-free DMPC liposome at the same DiR dose, confirming the higher brain-targeting efficiency of ANC compared with liposome (Figure 4). To see if ANC can be delivered into the brain parenchyma following intravenous administration,

vascular

visualization

was

performed

by

combining

immunofluorescence technique with confocal microscopy analysis using CD31 as the marker of blood vessels. Most of ANC in the brain was found to be able to penetrate across the BBB and distribute into the brain parenchyma (Figure 5). Following the delivery into the brain parenchyma, in both the cortex and hippocampus, the major site for Aβ aggregation, DiI-labeled ANC was found to highly accumulate at the surrounding of Aβ aggregates (Figure 6), suggesting that ANC might serve as a promising nanocarrier for delivery of diagnostics/therapeutics that can interact with Aβ in the CNS. To evaluate the efficiency of ANC in enhancing anti-AD drug delivery and the effect of ANC-α-M in alleviating the progression of AD pathology, SAMP8 mouse was used as the animal model. Anti-Aβ immunostaining analysis showed that following 2-week treatment of ANC-α-M, the level of amyloid plaque in the brains of SAMP8 mice were markedly decreased, compared with that in the NS, α-M solution or ANC-treated ones. Previous studies suggested that both Aβ oligomers and fibrils triggered neuroinflammatory cascades.33 We also found that compared with that in the brains of those animals given with saline, α-M solution and blank ANC, the number of CD45-positive activated microglia was much less in the ANC-α-M-treated SAMP8 mice (Figure 9). Given the central role of Aβ aggregates in the activation of microglia seen in AD brain and in AD animal models, the significant lessening in activated


Page 25 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


microglia found in the ANC-α-M-treated mice can be contributed by the improved drug delivery by ANC which enabled more efficient Aβ clearance. Such effect of ANC-α-M on lightening the pathological changes in AD model mice resulted in its best efficacy in rescuing memory deficits as the ANC-α-M-treated mice exhibited the most significant improvement in spatial and learning memory in the MWM task. Taken together, here we constructed a biologically-inspired nanocarrier which possesses both BBB permeability and high Aβ-binding affinity for the intervention of AD. The brain delivery efficiency and Aβ-targeting ability of the nanocarrier was confirmed both in vitro and in vivo. Following the incorporation of α-M, a polyphenolic agent, the Aβ-binding affinity of the nanoformulation was not reduced but

even

enhanced.

Also,

the

effect

of

ANC-α-M

on

facilitating

the

microglia-mediated uptake and degradation of Aβ1-42, decreasing amyloid deposition, attenuating microgliosis and rescuing memory impairment in an AD mouse model, was demonstrated. The findings of this work collectively suggested that the ApoE-based biomimetic nanocarrier might provide a promising platform for brain drug delivery towards the therapy of AD.

ASSOCIATED CONTENT Supporting Information Particle Size and Zeta Potential of DiO-labeled ANC and DiO-labeled ANC-α-M； Colocalization between DiO-labeled ANC/DiO-labeled ANC-α-M and lysosome after 3 h incubation.

AUTHOR INFORMATION Corresponding Authors *Address: Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, PR China. Tel: 86-21-63846590-776945. Fax: 86-21-64674721. E-mail: [email protected] (X.-L. G.). [email protected] (H.-Z. C.).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Author Contributions †

Q.-X. S. and H.-H. S. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was supported by National Natural Science Foundation of China (No. 81373351, 81573382), grants from Shanghai Science and Technology Committee (15540723700, 14ZR1423700), “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (15SG14) and funding from Shanghai Jiao Tong University (YG2014MS75). We would like to thank the National Center for Protein Science Shanghai for Cryo-EM analysis and Dr. Zhenglin Fu for his help in the preparation of Cryo-EM samples. REFERENCES: (1) Prince, M.; Wimo, A.; Guerchet, M.; Ali, G.; Wu, Y.; Prina, M. World Alzheimer Report 2015. 2015. (2) Jack, C. J.; Knopman, D. S.; Jagust, W. J.; Shaw, L. M.; Aisen, P. S.; Weiner, M. W.; Petersen, R. C.; Trojanowski, J. Q. Hypothetical Model of Dynamic Biomarkers of the Alzheimer's Pathological Cascade. Lancet Neurol. 2010, 9 (1), 119-128. (3) Ahmed, M.; Davis, J.; Aucoin, D.; Sato, T.; Ahuja, S.; Aimoto, S.; Elliott, J. I.; Van Nostrand, W. E.; Smith, S. O. Structural Conversion of Neurotoxic Amyloid-Beta(1-42) Oligomers to Fibrils. Nat. Struct. Mol. Biol. 2010, 17 (5), 561-567. (4) Viola, K. L.; Klein, W. L. Amyloid Beta Oligomers in Alzheimer's Disease Pathogenesis, Treatment, and Diagnosis. Acta Neuropathol. 2015, 129 (2), 183-206. (5) Canovi, M.; Markoutsa, E.; Lazar, A. N.; Pampalakis, G.; Clemente, C.; Re, F.; Sesana, S.; Masserini, M.; Salmona, M.; Duyckaerts, C.; Flores, O.; Gobbi, M.; Antimisiaris, S. G. The Binding Affinity of anti-Abeta1-42 MAb-decorated Nanoliposomes to Abeta1-42 Peptides in Vitro and to Amyloid Deposits in Post-Mortem Tissue. Biomaterials 2011, 32 (23), 5489-5497. (6) Mourtas, S.; Canovi, M.; Zona, C.; Aurilia, D.; Niarakis, A.; La Ferla, B.; Salmona, M.; Nicotra, F.; Gobbi, M.; Antimisiaris, S. G. Curcumin-Decorated Nanoliposomes with Very High Affinity for Amyloid-Beta1-42 Peptide. Biomaterials 2011, 32 (6), 1635-1645. (7) Cheng, K. K.; Chan, P. S.; Fan, S.; Kwan, S. M.; Yeung, K. L.; Wang, Y. X.; Chow, A. H.; Wu, E. X.; Baum, L. Curcumin-Conjugated Magnetic Nanoparticles for Detecting Amyloid Plaques in Alzheimer's Disease Mice Using Magnetic Resonance Imaging (MRI). Biomaterials 2015, 44, 155-172. (8) Brambilla, D.; Verpillot, R.; Le Droumaguet, B.; Nicolas, J.; Taverna, M.; Kona, J.; Lettiero, B.; Hashemi, S. H.; De Kimpe, L.; Canovi, M.; Gobbi, M.; Nicolas, V.; Scheper, W.; Moghimi, S. M.;


Page 26 of 29

Page 27 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Tvaroska, I.; Couvreur, P.; Andrieux, K. PEGylated Nanoparticles Bind to and Alter Amyloid-Beta Peptide Conformation: Toward Engineering of Functional Nanomedicines for Alzheimer's Disease. ACS Nano 2012, 6 (7), 5897-5908. (9) Gobbi, M.; Re, F.; Canovi, M.; Beeg, M.; Gregori, M.; Sesana, S.; Sonnino, S.; Brogioli, D.; Musicanti, C.; Gasco, P.; Salmona, M.; Masserini, M. E. Lipid-Based Nanoparticles with High Binding Affinity for Amyloid-Beta1-42 Peptide. Biomaterials 2010, 31 (25), 6519-6529. (10) Brambilla, D.; Le Droumaguet, B.; Nicolas, J.; Hashemi, S. H.; Wu, L. P.; Moghimi, S. M.; Couvreur, P.; Andrieux, K. Nanotechnologies for Alzheimer's Disease: Diagnosis, Therapy, and Safety Issues. Nanomedicine-UK 2011, 7 (5), 521-540. (11) Song, Q. X.; Huang, M.; Yao, L.; Wang, X. L.; Gu, X.; Chen, J.; Chen, J.; Huang, J. L.; Hu, Q. Y.; Kang, T.; Rong, Z. X.; Qi, H.; Zheng, G.; Chen, H. Z.; Gao, X. L. Lipoprotein-Based Nanoparticles Rescue the Memory Loss of Mice with Alzheimer's Disease by Accelerating the Clearance of Amyloid-Beta. ACS Nano 2014, 8 (3), 2345-2359. (12) Zhang, C.; Zheng, X. Y.; Wan, X.; Shao, X. Y.; Liu, Q. F.; Zhang, Z. M.; Zhang, Q. Z. The Potential Use of H102 Peptide-Loaded Dual-Functional Nanoparticles in the Treatment of Alzheimer's Disease. J. Control. Release 2014, 192, 317-324. (13) Lund-Katz, S.; Phillips, M. C. High Density Lipoprotein Structure-Function and Role in Reverse Cholesterol Transport. Subcell Biochem 2010, 51, 183-227. (14) Hauser, P. S.; Narayanaswami, V.; Ryan, R. O. Apolipoprotein E: From Lipid Transport to Neurobiology. Prog. Lipid Res. 2011, 50 (1), 62-74. (15) Holtzman, D. M.; Herz, J.; Bu, G. Apolipoprotein E and Apolipoprotein E Receptors: Normal Biology and Roles in Alzheimer Disease. Cold Spring Harb. Perspect. Med. 2012, 2 (3), a6312. (16) Patil, S. P.; Ballard, R.; Sanchez, S.; Osborn, J.; Santangelo, D. J. ApoE: The Link Between Alzheimer's-related Glucose Hypometabolism and Abeta Deposition? Med. Hypotheses 2012, 78 (4), 494-496. (17) Thal, D. R.; Capetillo-Zarate, E.; Schultz, C.; Rub, U.; Saido, T. C.; Yamaguchi, H.; Haass, C.; Griffin, W. S.; Del, T. K.; Braak, H.; Ghebremedhin, E. Apolipoprotein E Co-Localizes with Newly Formed Amyloid Beta-Protein (Abeta) Deposits Lacking Immunoreactivity Against N-terminal Epitopes of Abeta in a Genotype-Dependent Manner. Acta Neuropathol. 2005, 110 (5), 459-471. (18) Zensi, A.; Begley, D.; Pontikis, C.; Legros, C.; Mihoreanu, L.; Wagner, S.; Buchel, C.; von Briesen, H.; Kreuter, J. Albumin Nanoparticles Targeted with Apo E Enter the CNS by Transcytosis and are Delivered to Neurones. J. Control. Release 2009, 137 (1), 78-86. (19) LaDu, M. J.; Gilligan, S. M.; Lukens, J. R.; Cabana, V. G.; Reardon, C. A.; Van Eldik, L. J.; Holtzman, D. M. Nascent Astrocyte Particles Differ From Lipoproteins in CSF. J. Neurochem. 1998, 70 (5), 2070-2081. (20) Kuai, R.; Li, D.; Chen, Y. E.; Moon, J. J.; Schwendeman, A. High-Density Lipoproteins: Nature's Multifunctional Nanoparticles. ACS Nano 2016, 10 (3), 3015-3041. (21) Bricarello, D. A.; Smilowitz, J. T.; Zivkovic, A. M.; German, J. B.; Parikh, A. N. Reconstituted Lipoprotein: A Versatile Class of Biologically-Inspired Nanostructures. ACS Nano 2011, 5 (1), 42-57. (22) Yao, L.; Gu, X.; Song, Q. X.; Wang, X. L.; Huang, M.; Hu, M.; Hou, L. N.; Kang, T.; Chen, J.; Chen, H. Z.; Gao, X. L. Nanoformulated Alpha-Mangostin Ameliorates Alzheimer's Disease Neuropathology by Elevating LDLR Expression and Accelerating Amyloid-Beta Clearance. J. Control. Release 2016, 226, 1-14. (23) Wang, Y.; Xia, Z.; Xu, J. R.; Wang, Y. X.; Hou, L. N.; Qiu, Y.; Chen, H. Z. Alpha-Mangostin, a



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Polyphenolic Xanthone Derivative From Mangosteen, Attenuates Beta-Amyloid Oligomers-Induced Neurotoxicity by Inhibiting Amyloid Aggregation. Neuropharmacology 2012, 62 (2), 871-881. (24) Li, L.; Brunner, I.; Han, A. R.; Hamburger, M.; Kinghorn, A. D.; Frye, R.; Butterweck, V. Pharmacokinetics of Alpha-Mangostin in Rats After Intravenous and Oral Application. Mol. Nutr. Food Res. 2011, 55 Suppl 1, S67-S74. (25) Hu, Q. Y.; Gao, X. L.; Gu, G. Z.; Kang, T.; Tu, Y. F.; Liu, Z. Q.; Song, Q. X.; Yao, L.; Pang, Z. Q.; Jiang, X. G.; Chen, H. Z.; Chen, J. Glioma Therapy Using Tumor Homing and Penetrating Peptide-Functionalized PEG-PLA Nanoparticles Loaded with Paclitaxel. Biomaterials 2013, 34 (22), 5640-5650. (26) Huang, M.; Hu, M.; Song, Q. X.; Song, H. H.; Huang, J. L.; Gu, X.; Wang, X. L.; Chen, J.; Kang, T.; Feng, X. Y.; Jiang, D.; Zheng, G.; Chen, H. Z.; Gao, X. L. GM1-Modified Lipoprotein-Like Nanoparticle: Multifunctional Nanoplatform for the Combination Therapy of Alzheimer's Disease. ACS Nano 2015, 9 (11), 10801-10816. (27) Zhang, Z. H.; Cao, W. G.; Jin, H. L.; Lovell, J. F.; Yang, M.; Ding, L. L.; Chen, J.; Corbin, I.; Luo, Q. M.; Zheng, G. Biomimetic Nanocarrier for Direct Cytosolic Drug Delivery. Angew. Chem. Int. Ed. Engl. 2009, 48 (48), 9171-9175. (28) Butterfield, D. A.; Poon, H. F. The Senescence-Accelerated Prone Mouse (SAMP8): A Model of Age-Related Cognitive Decline with Relevance to Alterations of the Gene Expression and Protein Abnormalities in Alzheimer's Disease. Exp. Gerontol. 2005, 40 (10), 774-783. (29) Gilbert, B. J. The Role of Amyloid Beta in the Pathogenesis of Alzheimer's Disease. J. Clin. Pathol. 2013, 66 (5), 362-366. (30) Huang, Y.; Mucke, L. Alzheimer Mechanisms and Therapeutic Strategies. Cell 2012, 148 (6), 1204-1222. (31) Schnabel, J. Amyloid: Little Proteins, Big Clues. Nature 2011, 475 (7355), S12-S14. (32) Hamilton, R. L.; Williams, M. C.; Fielding, C. J.; Havel, R. J. Discoidal Bilayer Structure of Nascent High Density Lipoproteins From Perfused Rat Liver. J. Clin. Invest. 1976, 58 (3), 667-680. (33) Sondag, C. M.; Dhawan, G.; Combs, C. K. Beta Amyloid Oligomers and Fibrils Stimulate Differential Activation of Primary Microglia. J Neuroinflammation 2009, 6, 1.

ABSTRACT GRAPHIC


Page 28 of 29

Page 29 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents Only 88x38mm (280 x 280 DPI)


Biomimetic ApoE-Reconstituted High Density Lipoprotein Nanocarrier

Recommend Documents