Protection against Neurodegeneration in the Hippocampus Using

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/abseba

Protection against Neurodegeneration in the Hippocampus Using Sialic Acid- and 5‑HT-Moduline-Conjugated Lipopolymer Nanoparticles Jen-Tsung Yang,†,‡ Yung-Chih Kuo,*,§ I-Yin Chen,§ Rajendiran Rajesh,§ Yung-I Lou,∥ and Jyh-Ping Hsu⊥ †

Department of Neurosurgery, Chang Gung Memorial Hospital, 6, West Sec., Chia-Pu Road, Chia-Yi, Taiwan 61363, ROC College of Medicine, Chang Gung University, 259, Wenhua First Road, Tao-Yuan, Taiwan 33302, ROC § Department of Chemical Engineering, National Chung Cheng University, 168, University Road, Chia-Yi, Taiwan 62102, ROC ∥ Department of Accounting, Providence University, 200, Taiwan Boulevard, Taichung, Taiwan 43301, ROC ⊥ Department of Chemical Engineering, National Taiwan University, 1, Sec. 4, Roosevelt Road, Taipei, Taiwan 10617, ROC

ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by WEBSTER UNIV on 03/04/19. For personal use only.

‡

ABSTRACT: Significant involvement of oxidative stress in the brain can develop Alzheimer’s disease (AD); however, a great number of clinical trials explains the limited success of antioxidant therapy in dealing with this neurodegenerative disease. Here, we established a lipopolymer system of poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) incorporated with phosphatidic acid (PA) and modified with sialic acid (SA) and 5-hydroxytryptamine-moduline (5HTM) to improve quercetin (QU) activity against oxidative stress induced by amyloid-β (Aβ) deposits. Morphological studies revealed a uniform exterior of QU-SA-5HTM-PA-PLGA NPs with a spherical structure and enhanced aggregation with inclusion of PA in the formulation. A better brain-targeted delivery of the lipopolymeric NPs was verified from the high blood−brain barrier (BBB) permeability of QU through strong interactions of surface SA and 5HTM with Olinked N-acetylglucosamine and 5-HT1B receptors, respectively. Immunofluorescence staining images also supported QU-SA5HTM-PA-PLGA NPs to traverse the microvessels of AD rat brain. Western blot analysis showed that QU-loaded PA-PLGA NPs suppressed caspase-3 expression. The ability of the nanocarriers to recognize Aβ fibrils was demonstrated from the reduced senile plaque formation and the attenuated acetylcholinesterase and malondialdehyde activity in the hippocampus. Hence, the medication of QU-SA-5HTM-PA-PLGA NPs can facilitate the BBB penetration and prevent Aβ accumulation, lipid peroxidation, and neuronal apoptosis for the AD management. KEYWORDS: lipopolymer, poly(lactide-co-glycolide), phosphatidic acid, sialic acid, 5-HT-moduline

■

neurons, (ii) Aβ deposition fluctuates with the source of amyloid-containing biological substance, (iii) the size and composition of neuritic plaques depend on the morphology and growth condition of Aβ fibrils, and (iv) the resistance to drugs that inhibit Aβ accumulation varies between patients with mildly and rapidly progressive AD.3−5 Aβ fibrils and protofibrils, as well as Aβ oligomers were assumed to be the reason for Aβ neurotoxicity. A few recent studies have also reported that Zn, Cu, and Fe were involved in the AD pathogenesis either by direct interaction with Aβ to enhance its aggregation or by expediting the production of reactive oxygen species (ROS).6 Pharmaceutical industries have developed drugs targeting several mechanistic pathways to prevent Aβ

INTRODUCTION

Alzheimer’s disease (AD), a wide-spreading brain-degenerative disease, is evidenced from the appearance of senile plaques from amyloid-β (Aβ) aggregation and neurofibrillary lesions from tau protein entanglement, resulting in neuronal loss in the cerebral cortex. Apart from that, alteration in cholinergic neurotransmission, structural and functional irregularity in the mitochondria, synapse loss, oxidative stress, and inflammatory response are also associated with AD. A multiplicity of factors, including genetic and environmental, have been implicated in the AD pathogenesis.1,2 Genetic researches of all known forms of familial AD indicate a leading role for Aβ. However, most of the therapies aimed at reducing Aβ in the brain have failed due to the unavailability of a certified molecular mechanism for Aβ neurotoxicity. Further, Aβ is complex, and this complexity arises from the following factors: (i) different molecular structures of Aβ exhibit different levels of cytotoxicity to © XXXX American Chemical Society

Received: October 28, 2018 Accepted: February 19, 2019 Published: February 19, 2019 A

DOI: 10.1021/acsbiomaterials.8b01334 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

Figure 1. Physicochemical properties of QU-SA-5HTM-PA-PLGA NPs. (A) Schematic illustration; (B) particle size (Δ) and zeta potential (○), n = 3; (C) SEM and TEM images, PPA = 5%, (a)−(c) SEM, (d)−(f) TEM, (a) and (d) QU-PLGA NPs, (b) and (e) QU-SA-5HTM-PA-PLGA NPs, (c) and (f) close-up of a QU-SA-5HTM-PA-PLGA NP; (D) grafting efficiency of 5HTM (Δ) and SA (○) on QU-PA-PLGA NPs, PPA = 5%, n = 3.

generation, but they were unsuccessful.7 Among the 244 drugs aimed at treating AD that have undergone clinical trials from 2002 to 2012, only one has obtained the final approval from the United States Food and Drug Administration (U.S. FDA).8 Nevertheless, materials carrying drugs that target Aβ and ROS have been the subject of increasing interest in an effort to advance the efficacy against neuritic plaque formation. Lipopolymer used as a drug delivery system (DDS), with components made up of polymer and lipid, has higher stability than liposomes in physiological fluids and enhances targeted delivery to specific cell populations.9 For example, polyethylenimine-methoxy(polyethylene glycol) (PEG)-cholesterol lipopolymer facilitated the delivery of CRISPR/Cas9.10 Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) is a nanosized biopolymer approved by the U.S. FDA and is degradable via hydrolysis of its ester linkages. However, PLGA NPs as a DDS has certain limitations, such as randomized and concentration-dependent profile for biodistribution and pharmacokinetics, robust accumulation in tissues, and immediate degradation in the initial stage.11,12 To overcome these drawbacks, particulate PLGA may require modification to ameliorate its stability and blood circulation half-life. Our previous study supported the role of 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000] (DSPE-PEG(2000)-COOH) and didodecyldimethylammonium bromide in stabilizing colloidal PLGA particles.13 Phosphatidic acid (PA), a charged phospholipid, has the potential to interact with Aβ deposits and contributes to the

stability of the cell membrane.14 Hence, incorporation of PA into PLGA NPs may reduce Aβ aggregation in the cerebrum to attenuate the progression of AD. The blood−brain barrier (BBB), consisting mainly of specialized endothelia, represents the interface between neural and cardiovascular system and acts as an entrance shield for the selective transport of substances into the central nervous system (CNS).15,16 Surface modification of a DDS with sialic acid (SA) could facilitate the permeation across the BBB via receptor-mediated endocytosis (RME) using sialoadhesins that were found on endothelial cells (ECs).17 Modification with 5hydroxytryptamine-moduline (5HTM) could also help a DDS recognize brain microvascular ECs.18 5HTM controls serotonergic activity through regulating 5-HT1B receptors, which can modify neuronal behavior for distinct cerebral functions and retards neurodegeneration via suppressing interleukin-6 and tumor necrosis factor-α expression.19,20 The goal of this study was to develop lipopolymer drug carriers to circumvent the disadvantage of the low stability and nonspecific tissue intake of conventional polymeric and liposomal preparations and improve conjugation with Aβ fibrils. We constructed a hybrid lipopolymer system containing PLGA and PA to carry quercetin (QU). QU is a nonenzymatic antioxidant that counteracts oxidative stress and can reduce extracellular β-amyloidosis, tauopathy, astrogliosis, and microgliosis.21 QU-PA-PLGA NPs were modified with SA and 5HTM as multiresponsive nanocarriers for penetrating the B


Article

ACS Biomaterials Science & Engineering BBB and targeting fibrillar Aβ, and their efficacy in protecting AD brains was assessed.

■

Transendothelial Electrical Resistance (TEER), Permeability of PI, and Delivery of QU-SA-5HTM-PA-PLGA NPs across the BBB. A BBB model (HBMEC/HA/HBVP) was established using HBMECs, HAs (Sciencell), and HBVPs (Sciencell) cocultured on PET membrane (PETM) as per the literature protocal.22 The upper chamber of the transwell after treatment with QU-SA-5HTM-PAPLGA NPs (0.025% (w/v)) for 24 h was used to measure the TEER (Ω × cm2) with the help of an electrical resistance device (Millicell, Millipore). The TEER was calculated by [(electrical resistance of PETM containing BBB cells−electrical resistance of intact PETM) × area of PETM]. The permeability of propidium iodide (PI; Sigmaaldrich) across the BBB was evaluated by treating PI (0.025% (w/v)) in the upper chamber after being affected by QU-SA-5HTM-PAPLGA NPs for 4 h. The permeated PI in the lower chamber was determined with the help of an ELISA spectrofluorometer at excitation and emission wavelengths of 485 and 590 nm, respectively. The permeability of PI, PPI, was calculated by [(permeation of PI across PETM containing BBB cells)−1 − (permeation of PI across intact PETM)−1]−1. The permeability of QU, PQU, was examined by treating QU-SA-5HTM-PA-PLGA NPs (0.025% (w/v)) in the upper chamber. From that, the liquid in the lower chamber (20 μL) was taken out every 2.5 h and subjected to the HPLC-UV setup to measure the amount of QU. PQU was calculated by an equation similar to the equation for calculating PPI. Immunochemical Stains of HBMECs and SK-N-MC Cells Insulted with Aβ and Treated with QU-SA-5HTM-PA-PLGA NPs. HBMECs (1 × 105 cells/cm2) were cultured on a gelatinpretreated coverslip in a tissue culture plate (24-well) for 8 h, followed by treatment with fluorescent QU-SA-5HTM-PA-PLGA NPs (0.0025% (w/v)) in the medium for 1.5 h. Then, the cultured cells were reacted with formalin (10% (v/v), Sigma-Aldrich), and carried out a series of treatment with Triton-X-100 (0.5% (v/v), Acros Organics), blocking reagent (casein-based (CAS), Invitrogen) for 30 min, diluted primary antibody of anti-O-linked N-acetylglucosamine (1:100, Abcam) and/or guinea pig anti-5-HT1B (1:300, Chemicon) at 4 °C overnight. Primary antibody-treated HBMECs were treated with diluted secondary antibody of rhodamineconjugated goat antimouse immunoglobulin G (IgG; heavy and light chains (H+L), 1:300, Abcam) for 1 h, followed by reaction with 4′,6-diamidino-2-phenylindole (DAPI; 0.5% (w/v), Sigma-Aldrich). A confocal laser scanning microscope (CLSM; Zeiss) was utilized to obtain the immunochemical staining images at excitation and emission wavelengths of 350 nm (DAPI), 490 nm (FITC) and 540 nm (rhodamine), and 475 nm (DAPI), 520 nm (FITC) and 565 nm (rhodamine), respectively. The procedures to prepare the sample for staining cultured SK-NMC cells and to study immunostaining results were similar to the procedures for HBMECs. SK-N-MC cell and HBMEC systems were different from the use of Aβ1−42 (10 μM) with fluorescent QU-SA5HTM-PA-PLGA NPs and the use of anti-Aβ antibody (monoclonal, 1:400, Abcam) for the former. In the case of caspase-3 expression, anti-Aβ antibody was replaced by anticaspase-3 (1:400, Millipore). Western Blot of Caspase-3 after Treatment with QU-SA5HTM-PA-PLGA NPs. SK-N-MC cells (1 × 105 cells/well) were cultured in a gelatin-pretreated tissue culture plate (12-well), followed by treatment with Aβ1−42 (10 μM) and QU-SA-5HTM-PA-PLGA NPs, and addition of lysis buffer (200 μL/well, Abcam). From this, lysed protein solution (150 μL, 1% (v/v)) was treated with a CBK (150 μL) to measure the total protein with the help of an ELISA spectrofluorometer at a wavelength of 595 nm. After that, the sample (25 μg of total protein) was mixed with sample buffer (25% (v/v), Millipore) at 95 °C. The process for the migration of protein in separating gel was done at two increasing voltage levels, and then relocated to polyvinylidene fluoride (GE Healthcare) membrane. Further, the polyvinylidene fluoride membrane containing protein was incubated with skim milk (5% (w/v)), treated with diluted primary antibody of anticaspase-3 (1:1000) and antiprocaspase-3 (1:1000, Millipore), followed by treatment with diluted secondary antibody of goat antirabbit IgG (H+L, horseradish peroxidase, 1:2000, Abcam). Finally, polyvinylidene fluoride membrane containing protein was

MATERIALS AND METHODS

Fabrication of QU-SA-5HTM-PA-PLGA NPs. PLGA (8−10 mg, Purac), 1,2-dipalmitoyl-sn-glycero-3-phosphate (0−1 mg, Avanti Polar Lipids), DSPE-PEG(2000)-COOH (0.5 mg, Avanti Polar Lipids) and QU (0.5 mg, Sigma-Aldrich) were added in chloroform (5 mL, J.T. Baker) and stirred for 1 h. Fluorescein isothiocyanate (FITC; 50 ppm, Sigma-Aldrich) was added to the organic mixture for preparing fluorescent NPs. Emulsification of the solution was carried out at 25 °C for 20 min using Tween 80 (50 mL, 0.4% (w/v), Fisher Scientific) with homogenization at 22 000 rpm. Ultrapure water (UW; 140 mL) was mixed with the resultant mixture and stirred for 1 h, followed by filtration and high-speed centrifugation. The supernatant was subjected to high-performance liquid chromatography (HPLC; Jasco), followed by UV−visible detection (370 nm), using methanol (50% (v/v), Mallinckrodt Baker) in UW as mobile phase (1 mL/min) to determine the concentration of loaded QU in PLGA NPs. The loading of QU in QU-PA-PLGA NPs was evaluated on the basis of the total weight of QU used in the preparation. QU-PA-PLGA NPs were resuspended in a dialysis tube (Spectrum Laboratories) and dialyzed in UW (200 mL) for 1 h, followed by centrifugation, airdrying, freezing, and lyophilization. QU-PA-PLGA NPs (1 mg/mL) were stirred with SA (25 μg/mL, Sigma-Aldrich) and/or 5HTM (25 μg/mL, Kelowna International Scientific), N-hydroxysuccinimide (NHS; 0.1 mg/mL, Acros), and 1ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC; 0.2 mg/mL, Sigma-Aldrich) at 80 rpm and 4 °C overnight, followed by centrifugation to obtain QU-SA-5HTM-PA-PLGA NPs (Figure 1A). The supernatant was mixed with o-phenylenediamine (20 mg/mL, Sigma-Aldrich) for 30 min, and the amount of SA was examined with the help of an enzyme-linked immunosorbent assay (ELISA) spectrofluorometer at excitation and emission wavelengths of 230 and 425 nm, respectively. The amount of 5HTM in the supernatant was detected using a Coomassie blue kit (CBK; Thermo Fisher) and examined with the help of an ELISA spectrofluorometer at a wavelength of 595 nm. The cross-linking of SA and 5HTM on QUPA-PLGA NPs, ESA (%) and E5HTM (%), was quantified by [(total weight of SA or 5HTM − unloaded weight of SA or 5HTM)/(total weight of SA or 5HTM)] × 100%. Characterization of QU-SA-5HTM-PA-PLGA NPs. The particle size, D, and zeta potential, ζ, of drug-loaded PLGA NPs were examined by mixing the drug carriers (2 mg/mL) in Tris buffer (0.1 M, Riedel-de Haen) and measured with the help of zetasizer 3000 HSA (Malvern) for 20 s. The sample of QU-SA-5HTM-PA-PLGA NPs was dehydrated, platinum-coated, and subjected to a field emission scanning electron microscope (FE-SEM; Jeol) at 3 kV for 90 s to visualize the surface geometry. The sample was pretreated with phosphotungstic acid (10 μL, 2% (w/v), Sigma-Aldrich) and subjected to a transmission electron microscope (TEM, Jeol) to visualize the particle structure. Cytotoxicity of QU-SA-5HTM-PA-PLGA NPs. Human brain microvascular ECs (HBMECs; 5 × 103 cells/well, Biocompare) or SK-N-MC cells (5 × 103 cells/well, American Type Tissue Collection) were cultured on a gelatin (20 mg/mL, Sigma-Aldrich)pretreated plate (96-well, polystyrene) in a CO 2 incubator (humidified, NuAire). Aβ1−42 (10 μM, Life Technologies) fibrils were mixed with medium to culture SK-N-MC cells. Then, the cultured cells were treated with 0.025% (w/v) QU-SA-5HTM-PAPLGA NPs for 12 h, followed by reaction with 2,3-bis(2-methoxy-4nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; 50 μL, 2% (v/v), Biological Industries) for 4 h. Finally, the optical density (OD) of XTT-treated cell suspension was measured with the help of an ELISA spectrofluorometer at a wavelength of 450 nm. The viability of HBMECs, PCV,HBMEC, and that of SK-N-MC cells, PCV,SK‑N‑MC, were evaluated by [(OD of cell culture with QU-SA-5HTM-PA-PLGA NPs − OD of XTT)/(OD of cell culture without QU-SA-5HTM-PAPLGA NPs − OD of XTT)] × 100%. C


Article


Figure 2. Effect of QU-SA-5HTM-PA-PLGA NPs on the BBB, PPA = 5%. (A) TEER and BBB penetration ability of PI, (I) control, (II) QU-PAPLGA NPs, (III) QU-SA-PA-PLGA NPs, (IV) QU-5HTM-PA-PLGA NPs, (V) QU-SA-5HTM-PA-PLGA NPs, n = 3; (B) viability of HBMECs, #: p > 0.05, n = 3; (C) BBB permeability of QU, *: p < 0.05, **: p < 0.01, n = 3; (D) immunochemical staining images, (a) control (no NPs), (b) QU-SA-PA-PLGA NPs, (c) QU-5HTM-PA-PLGA NPs, (d) QU-SA-5HTM-PA-PLGA NPs, (*-1) blue: nuclei, (*-2) green: nanocarriers, (a-3) and (d-3) red: O-linked N-acetylglucosamine and 5-HT1B receptors, (b-3) red: O-linked N-acetylglucosamine, (c-3) red: 5-HT1B receptors, (*-4) merged image, bar = 40 μm. brain was solidified, followed by fixing and freezing. Using a cryostat microtome (Leica), CA1 tissue in the hippocampus was sectioned into 10-μm samples at −16 °C to −20 °C. The sliced section was soaked in acetone (Mallinckrodt Baker), treated with hydrogen peroxide (0.1% (v/v), Sigma-Aldrich), fixed with 10% formalin, incubated with CAS blocking reagent for 1 h, and treated with antialpha-smooth muscle actin antibody (1:200, 50 μL, Millipore) or anti-O-linked N-acetylglucosamine antibody (1:100) and guinea pig anti-5-HT1B (1:300). Tetramethylrhodamine (1:800, 50 μL, SigmaAldrich) for antialpha-smooth muscle actin antibody and goat antirabbit IgG (1:800) for anti-O-linked N-acetylglucosamine antibody and guinea pig anti-5-HT1B were added to the sliced section at 25 °C for 1 h, followed by the addition of DAPI (5 μg/mL) in Triton X-100 (0.1% (v/v)). The stained section was sealed with a coverslip and analyzed using a CLSM. To analyze the Aβ plaque formation in the CA1, sliced hippocampus was incubated with primary antibody of anti-Aβ (monoclonal) and secondary antibody of goat antirabbit IgG (1:200), and then treated with substrate of 3,3′-diaminobenzidine (Millipore). The images of Aβ deposits were obtained using a Nikon microscope. AChE and MDA Assay of the Hippocampus after Treatment with QU-SA-5HTM-PA-PLGA NPs. After euthanasia, decomposition of homogenized CA1 tissue (5 mg) was decomposed at 4 °C by reacting with Thermo Fisher reagent, including lysis buffer (100 μL), protein extraction solution (100 μL), and Halt cocktail (1 μL), followed by centrifugation. Acetylcholinesterase (AChE) activity, AAChE, was evaluated by reacting the supernatant (50 μL) at 25 °C with Abcam reagent, including acetylthiocholine (50 μL) in bovine serum albumin (50 μL, 0.1%), followed by analyzing with the help of an ELISA spectrofluorometer at a wavelength of 410 nm. For malondialdehyde (MDA) activity, AMDA, decomposition of homogenized CA1 tissue (10 mg) was carried out by reacting with Abcam reagent, including lysis buffer (300 μL) and butylated hydroxytoluene

subjected to select enhanced chemiluminescence (GE Healthcare) before using a Luminescence Image Analysis System (Fujifilm) to determine the protein amount with ImageJ (National Institutes of Health). Transport of QU-SA-5HTM-PA-PLGA NPs and Histochemical Staining of Aβ Plaques in AD Rat Brain. Eight-week old male Wistar rats (BioLasco) were nourished as per the Institutional Animal Care and Use Committee guidelines, which was also followed to carry out the in vivo studies using these Wistar rats. The Institutional Animal Care and Use Committee of National Chung Cheng University authorized the Affidavit of Approval of Animal Use Protocol. First, 7−10 Wistar rats were assigned to a group that was insulted with Aβ1−42 and treated with QU-SA-5HTM-PA-PLGA NPs. The animals were denied food for 12 h before the test, and administered with atropin (0.4 mg/mL, total quantity of 0.4 mg/kg, Hebei Depond Animal Health Technology) via intraperitoneal injection, followed by injecting anesthesia of sodium pentobarbital (60 mg/mL, total quantity of 60 mg/kg, Siegfried) via intraperitoneal injection. Aβ1−42 (3.3 mg/mL, total volume of 15 μL, 2 μL/min) was injected into the drilled dorsoventral position. The position of cornu ammonis area 1 (CA1) was obtained by subtracting 0.35 cm from the anteroposterior position, 0.2 cm from the mediolateral position, and 0.27 cm from the dorsoventral position. The head cuts were stretched. The experimental rats were housed for 2 weeks to establish an AD rat model. Before intravenous (IV) injection, the needles were exposed to UV, and then the needles and the skin surface for injection were sterilized with cotton-containing ethanol. In treating the AD rats, 2 mg/mL of particulate formulations were injected via the tail vein for 1 mL of total volume. After IV injection, ethanol-soaked sterile cotton was used for hemostasis of the injection area. The IV injection was carried out every 2 days, three times per day, before euthanasia. The AD rats were sacrificed after 1 h of administration of fluorescent QUSA-5HTM-PA-PLGA NPs, which were delivered to the BBB. The D


Article

ACS Biomaterials Science & Engineering (3 μL), followed by centrifugation with the condition of measuring AAChE. AMDA was evaluated by reacting the supernatant (50 μL) at 95 °C with thiobarbituric acid (150 μL) for 1 h, followed by analyzing with the help of an ELISA spectrofluorometer at a wavelength of 532 nm. Statistics. Mean ± standard deviation has been used to present the data, and a one-way analysis of variance with Tukey’s honestly significant difference test has been used to determine statistical significance.

QU-SA-5HTM-PA-PLGA NPs. The impact of nanocarriers on the in vitro HBMEC/HA/HBVP coculture system enhanced the permeability of PI owing to the leakage of the TJs. QU-SA5HTM-PA-PLGA NPs had an effect on EC tightness via combined effects of recognition of O-linked N-acetylglucosamine by SA and 5-HT1B receptors by 5HTM. However, QUSA-5HTM-PA-PLGA NPs exhibited a tolerable level of the TEER of about 270 Ω × cm2 and the permeability of PI of about 4.9 cm/s. The required TEER was found to be more than 120 Ω × cm2 to maintain structural tightness of the BBB for delivery of pharmaceutical components to the brain with safe penetration.28 Hence, the current values of the two parameters supported the enhancement in the transport across the BBB without imperiling structural integrity after treatment with QU-SA-5HTM-PA-PLGA NPs. Toxicity of QU-SA-5HTM-PA-PLGA NPs to HBMECs. The viability of HBMECs treated with QU-SA-5HTM-PA-PLGA NPs is shown in Figure 2B. As revealed in this figure, QU-SA5HTM-PA-PLGA NPs were slightly toxic to HBMECs. However, there was not much difference in the viability (∼90−95%) after treatment with different groups of QUloaded PA-PLGA NPs, which proved the biocompatibility of QU-SA-5HTM-PA-PLGA NPs. The toxicity of QU-SA5HTM-PA-PLGA NPs could be corroborated from the membrane fusion and subsequent transcellular penetration through HBMECs. NPs could also induce inflammatory factors to inhibit the cell proliferation.19,29,30 QU-SA-5HTM-PAPLGA NPs showed lower viability of HBMECs, compared with QU-SA-PA-PLGA NPs and QU-5HTM-PA-PLGA NPs. This could be attributed to SA and 5HTM activity with improved binding and increased membrane transfusion. Overall, QU-SA-5HTM-PA-PLGA NPs could be a targeted DDS with a permissible degree of toxicity with viability higher than 90% in the BBB cells. BBB Permeability of QU Using QU-SA-5HTM-PA-PLGA NPs. The capability of QU-SA-5HTM-PA-PLGA NPs to cross the BBB is shown in Figure 2C. The permeability of QU followed the order of QU-SA-5HTM-PA-PLGA NPs > QU5HTM-PA-PLGA NPs > QU-SA-PA-PLGA NPs > QU-PAPLGA NPs. Incorporation of SA and 5HTM into PA-PLGA NPs facilitated the BBB permeability of QU for brain-targeted therapy, better than the BBB permeability using QU-loaded PA-PLGA NPs, through regulating the intake via RME. Immunofluorescence Staining of HBMECs Treated with QU-SA-5HTM-PA-PLGA NPs. Figure 2D shows the fluorescence staining images of QU-SA-5HTM-PA-PLGA NPs internalized by endothelia through expressed O-linked Nacetylglucosamine and/or 5-HT1B receptors. The blue, green, and red stains indicate, respectively, HBMEC nuclei, fluorescent NP carriers, and membrane markers. Figure 2D(b) revealed a smaller number of QU-SA-PA-PLGA NPs on O-linked N-acetylglucosamine compared with QU-5HTMPA-PLGA NPs ((Figure 2D(c)) on 5-HT1B receptors. This demonstrated that 5HTM-grafted PA-PLGA NPs had a stronger effect on particle internalization by HBMECs than SA-grafted PA-PLGA NPs. Among all the groups, the strongest green intensity was obtained in the case of QU-SA-5HTM-PAPLGA NPs ((Figure 2D(d)). The immunofluorescence was consistent with Figure 2A−C and verified that the combination of surface SA and 5HTM activated the intake of PA-PLGA NPs to penetrate the BBB. Rescue of SK-N-MC Cells Insulted with Aβ Using QU-SA5HTM-PA-PLGA NPs. The Aβ-induced neurotoxicity to SK-N-

■

RESULTS AND DISCUSSION Characterizations of QU-SA-5HTM-PA-PLGA NPs. The impact of differenet amount of PA on the diameter and zeta potential of QU-SA-5HTM-PA-PLGA NPs is shown in Figure 1B. The particle diameter of QU-SA-5HTM-PA-PLGA NPs appeared to be in the range of 140−200 nm and increased with increases in the PA content. An increasing PA content increased the hydrophobicity of QU-SA-5HTM-PA-PLGA NPs and influenced the packing of surface molecules.23 Similarly, an increasing PA content increased the negative charge of QU-SA-5HTM-PA-PLGA NPs, leading to increased electrical repulsion among particles for better stability. The size of QU-SA-5HTM-PA-PLGA NPs was considered as one of the primary criteria, since it affected the blood clearance process. QU-SA-5HTM-PA-PLGA NPs with a smaller size might be removed at a lower mass-transfer efficiency than those with a larger size via extravasation.24 Hence, QU-SA-5HTM-PAPLGA NPs prepared with 5% PA were utilized for further experimental studies. The surface and structural morphology of PLGA NPs was visuvalized through SEM and TEM images (Figure 1C). These images revealed uniformly distributed QU-PLGA NPs with a regular spherical shape. The particle linkage tended to appear more obviously in QU-SA-5HTM-PA-PLGA NPs, which supported the increased hydrophobicity of QU-SA-5HTMPA-PLGA NPs with increases in the PA weight percentage. Further, the TEM images exhibited the appearance of SA, 5HTM and PA on the external layer of QU-SA-5HTM-PAPLGA NPs (Figure 1C(e,f)), which could ameliorate the stability of QU-SA-5HTM-PA-PLGA NPs and help distribute an amount of hydrophobic QU. Moreover, the hydrophilic headgroup of PA might be oriented randomly to stabilize the colloidal structure.25 SA and 5HTM were conjugated to the carboxyl group of DSPE-PEG(2000)-COOH on lipophilic PA-PLGA NPs by EDC/NHS coupling. The grafting efficiency of SA and 5HTM on PA-PLGA NPs decreased with increases in the concentration of SA and 5HTM (Figure 1D). A high concentration of SA and 5HTM resulted in strong competition for reaction sites on PA-PLGA NPs, and it reached a saturation level of the carboxyl group for further reaction. This led to unavailability of conjugation site on PA-PLGA NPs for SA and 5HTM and a reduced grafting efficiency. BBB Permeability using QU-SA-5HTM-PA-PLGA NPs. TEER and BBB Permeability of PI. Figure 2A shows the endothelial tightness indexes of the in vitro HBMEC/HA/ HBVP coculture system after the impact of various groups of lipopolymer NP formulation. The TEER provides information on the penetration ability through tight junctions (TJs) between endothelia; decreased TEER is expected during the temporary opening of TJs for a DDS to cross the BBB.26,27 Treatment with these NP carrier systems slightly disrupt TJ structure in the BBB, in the order of control > QU-PA-PLGA NPs > QU-SA-PA-PLGA NPs ∼ QU-5HTM-PA-PLGA NPs > E


Article


Figure 3. Effect of QU-SA-5HTM-PA-PLGA NPs on viability of and targeting to SK-N-MC cells insulted with Aβ for 24 h. (A) Cell viability, PPA = 5%, #: p > 0.05, *: p < 0.05, **: p < 0.01, n = 3; (B) immunochemical staining images, (a) QU-SA-5HTM-PLGA NPs, (b) QU-SA-5HTM-PAPLGA NPs, PPA = 1%, (c) QU-SA-5HTM-PA-PLGA NPs, PPA = 3%, (d) QU-SA-5HTM-PA-PLGA NPs, PPA = 5%, (*-1) blue: nuclei, (*-2) green: nanocarriers, (*-3) red: Aβ, (*-4) merge, bar = 100 μm.

green signal, and the green intensity was promoted with increases in the PA weight percentage from 1% to 5%, suggesting increased ability of the nanocarriers to target Aβ. The negatively charged PA had an electrical attraction to the positively charged residue on Aβ, which accelerated the adherence of QU-SA-5HTM-PA-PLGA NPs to Aβ.32,33 Therefore, QU-SA-5HTM-PA-PLGA NPs could target Aβ with a high affinity, to affect Aβ aggregation with focal release of QU. Caspase-3 Expression in SK-N-MC Cells Insulted with Aβ and Treated with QU-SA-5HTM-PA-PLGA NPs. Figure 4A shows the Western blots of expressed caspase-3 and its ratio to procaspase-3 in SK-N-MC cells insulted with Aβ and treated with QU, QU-SA-5HTM-PLGA NPs, and QU-SA-5HTM-PAPLGA NPs. The induction of apoptosis requires the activation of one or more members of the caspases, which needs to be downregulated to increase the cell viability.34 An insult with Aβ to SK-N-MC cells stimulated caspase-3 activity, and treatment with QU and its nanocarrier formulations downregulated caspase-3 expression. The relative ratio was in the order of QU-SA-5HTM-PA-PLGA NPs < QU-SA-5HTM-PLGA NPs < QU. QU alone did not have much of an impact on downregulating caspase-3 expression. QU-SA-5HTM-PLGA NPs exhibited a considerable reduction in caspase-3 expression. QU-SA-5HTM-PA-PLGA NPs revealed the lowest caspase-3 expression, almost equal to the caspase-3 expression of the control, owing to the combined ability of PA to target Aβ and QU to protect neurons. The results were consistent

MC cells under the influence of QU, QU-PLGA NPs, and QUSA-5HTM-PA-PLGA NPs is shown in Figure 3A. Treatment with Aβ fibrils inhibited proliferation and induced an obvious reduction in the cell viability. The toxicity of Aβ to SK-N-MC cells was substantially ameliorated via treatment with QU and its lipophilic formulation. The order of the viability appeared as QU-SA-5HTM-PA-PLGA NPs > QU-PLGA NPs > QU. This order can be elucidated as follows: (i) QU suppressed Aβinduced oxidative stress on apoptotic SK-N-MC cells and could potentiate the effects of glutathione peroxidase on the detoxification of ROS;31 (ii) QU-PLGA NPs reduced the release rate of QU and prolonged bioavailability of QU; (iii) PA enhanced the targeting to Aβ to increase the local concentration of QU around SK-N-MC cells; and (iv) surface PA, SA, and 5HTM ameliorated the stability of QU-PLGA NPs to increase the possibility of carrier internalization. As a result, QU-SA-5HTM-PA-PLGA NPs favored the topical delivery of QU to protect neurons from Aβ-induced degeneration and might stimulate cell proliferation. The targeting of QU-SA-5HTM-PA-PLGA NPs with various PA weight percentages to SK-N-MC cells surrounded by Aβ is shown in Figure 3B. These images depicted red Aβ accumulation near blue SK-N-MC cells. Further, the NPs (green stains) were attached to the surface of Aβ deposits. Figure 3B(a) indicated that QU-SA-5HTM-PLGA NPs had the weakest green intensity near the red stains, explaining the lowest targeting ability against Aβ. However, incorporation of PA into PLGA NPs (Figure 3B(b-d)) increased the intensity of F


Article


Figure 4. Caspase-3 expressed by SK-N-MC cells insulted with Aβ and treated with QU-SA-5HTM-PA-PLGA NPs for 24 h. (A) protein level of caspase-3, PPA = 5%, (a) Western blot, (b) ratio of caspase-3/pro-caspase; #: p > 0.05, *: p < 0.05, **: p < 0.01, n = 3; (B) fluorescent images of staining against caspase-3, (a) control, (b) Aβ, (c)−(e) with Aβ, (c) QU, (d) QU-SA-5HTM-PLGA NPs, (e) QU-SA-5HTM-PA-PLGA NPs, PPA = 5%, (*-1) blue: nuclei, (*-2) red: activated caspase-3, (*-3): merged image, bar = 10 μm.

physiological and functional regulation of pericytes in the NVU.35,36 As evidenced from the immunochemical staining of the BBB, the surface modification with SA and 5HTM was crucial to the ability of QU-SA-5HTM-PA-PLGA NPs to traverse the BBB via pericytes in AD rat brain. Immunohistochemistry of AD Rat Brain Treated with QUSA-5HTM-PA-PLGA NPs. The immunohistochemistry of Aβ plaques in AD brain tissue after treatment with QU, U-PLGA NPs and QU-SA-5HTM-PA-PLGA NPs is shown in Figure 5B. Treatment of the rat brain with Aβ fibrils (Figure 5B(b)) produced a high amount of neurotic plaques in the hippocampus. Free QU (Figure 5B(c)) exhibited the weakest effect against reducing Aβ deposition in the hippocampus. The deposition of Aβ plaques was also observed in the case of QUPLGA NPs (Figure 5B(d)). The image of the QU-SA-5HTMPA-PLGA NP group (Figure 5B(e) is almost the same as that of the sham group free from Aβ plaque formation, suggesting normal neural tissue. The following three reasons can explain the complete reduction of Aβ deposition by treatment with QU-SA-5HTM-PA-PLGA NPs. First, surface SA and 5HTM assisted in the access of particulate nanomaterials to traverse the BBB and increased the concentration of QU in the CNS to reduce Aβ fibril-induced oxidative stress. Second, 5HTM could upregulate dopamine activity.37 A recent survey found that dopaminergic dysfunction intensified neuronal degeneration, and that this is the reason for the symptoms of AD.38 Third, a

with the staining images of caspase-3 expressed by SK-N-MC cells insulted with Aβ and treated with the nanocarriers (Figure 4B). Treatment with Aβ (Figure 4B(a)) yielded a high caspase-3 expression (red stains) near the nuclei of SK-N-MC cells (blue stains). Immunofluorescence of caspase-3 disappeared in the same order as that of caspase-3 expressed in Western blots. Thus, QU-SA-5HTM-PA-PLGA NPs were of great impact on downregulating caspase-3 expression to protect Aβ-insulted neurons from degeneration. Distribution of QU-SA-5HTM-PA-PLGA NPs in the Microvessels of AD Rat Brain. The characteristics of the BBB are stimulated and maintained by crosstalk among ECs and other members of the neurovascular unit (NVU), in which pericytes appear closer to endothelia and contribute to the maturation of barrier tightness. Figure 5A shows the images of fluorescent QU-SA-5HTM-PA-PLGA NPs (green stains) distributed in the BBB of AD rats. The blue stains are the nuclei in brain tissue near the BBB in vivo. As indicated in Figure 5A(a), QUSA-5HTM-PA-PLGA NPs appeared around the α-smooth muscle composed of pericytes (red stains). This demonstrated the ability of these NP carriers to pass through the endothelial barricade via cerebral vascular smooth muscle. Further, Figure 5A(b) depicted a high quantity of QU-SA-5HTM-PA-PLGA NPs near the membrane containing O-linked N-acetylglucosamine and 5-HT1B receptors (red stains). Thus, RME of QUSA-5HTM-PA-PLGA NPs could take place through the G


Article


Figure 5. Neuroprotection of QU-SA-5HTM-PA-PLGA NPs against hippocampal degeneration in AD rats. (A) Transport across the BBB after intravenous administration for 1 h, (*-1) blue channel for nuclei, (*-2) green channel for QU-SA-5HTM-PA-PLGA NPs, (a-3) red channel for staining against alpha-smooth muscle, (b-3) red channel for staining against O-linked N-acetylglucosamine and 5-HT1B receptors, (*-4) merged image, (a) 630X, bar = 75 μm, (b) 1890X, bar = 25 μm; (B) immunostaining of Aβ deposition in brain tissue of 14-day Aβ-insulted Wistar rats, (a) sham, (b) Aβ, (c)−(e) with Aβ, (c) QU, (d) QU-PLGA NPs, (e) QU-SA-5HTM-PA-PLGA NPs, PPA = 5%, bar = 25 μm; (C) AChE and MDA activity of AD rat model for 14 days, PPA = 5%, #: p > 0.05, *: p < 0.05, **: p < 0.01, n = 5.

and its lipopolymer formulation. The order in the decrease in AChE and MDA activity was QU < QU-PLGA NPs < QU-SA5HTM-PA-PLGA NPs. This revealed that QU-SA-5HTM-PAPLGA NPs could assist in crossing the BBB, promote binding to Aβ, reduce the amount of Aβ plaques, and suppress neurotoxic oxidative stress. Hence, SA-5HTM-PA-PLGA NPs had an active role, along with QU, in protecting the hippocampus of the AD brain.

high affinity of PA to Aβ plaques caused the topical delivery of QU around degenerated neurons. AChE and MDA Activity in the CA1 of AD Rats after Treatment with QU-SA-5HTM-PA-PLGA NPs. Figure 5C shows the role of lipopolymer in the variation of neural chemistry in Aβ-insulted hippocampus. Aβ fibrils considerably increased AChE and MDA activity, suggesting a substantial reduction in acetylcholine activity and acceleration of lipid peroxidation in AD brain. Treatment with Aβ could inhibit the formation of neurotransmitter and induce neurodegeneration through production of ROS. Increasing AChE activity was associated with senile plaques through colocalization with Aβ deposits in the AD brain.39,40 An increased MDA level also indicated progressive stimulation of Aβ-induced oxidative stress.41,42 As indicated in Figure 5C, the increased AChE and MDA activity were attenuated by treatment with free QU

■

CONCLUSIONS PA-PLGA NPs were prepared and modified with SA and 5HTM. Conjugation of SA and 5HTM on the lipopolymer assisted in delivering QU with high BBB permeability and in internalizing QU-SA-5HTM-PA-PLGA NPs in the microvessels of AD rat brain. In addition, incorporation of PA into QU-SA-5HTM-PA-PLGA NPs increased the absolute value of H


Article

ACS Biomaterials Science & Engineering zeta potential, upgraded binding affinity to Aβ fibrils for better QU bioavailability, and prevented neurotoxicity induced from oxidative stress. Based on Western blot and immunohistochemistry analyses, QU-SA-5HTM-PA-PLGA NPs downregulated caspase-3 expression, reduced Aβ deposits, suppressed AChE and MDA activity in degenerated hippocampus, and enabled neuroprotection against AD pathology.

■

(11) Makadia, H. K.; Siegel, S. J. Poly lactic-co-glycolic acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers (Basel, Switz.) 2011, 3, 1377−1397. (12) Kuo, Y. C.; Tsai, H. C. Rosmarinic acid- and Curcumin-Loaded Polyacrylamide-Cardiolipin-Poly(lactide-co-glycolide) Nanoparticles with Conjugated 83−14 Monoclonal Antibody to Protect βAmyloid-Insulted Neurons. Mater. Sci. Eng., C 2018, 91, 445−457. (13) Kuo, Y. C.; Chen, Y. C. Targeting Delivery of Etoposide to Inhibit the Growth of Human Glioblastoma Multiforme Using Lactoferrin- and Folic acid-Grafted Poly(lactide-co-glycolide) Nanoparticles. Int. J. Pharm. 2015, 479, 138−149. (14) Re, F.; Cambianica, I.; Sesana, S.; Salvati, E.; Cagnotto, A.; Salmona, M.; Couraud, P.-O.; Moghimi, S. M.; Masserini, M.; Sancini, G. Functionalization with ApoE-derived Peptides Enhances the Interaction with Brain Capillary Endothelial Cells of Nanoliposomes Binding Amyloid-beta Peptide. J. Biotechnol. 2011, 156, 341−346. (15) Zenaro, E.; Piacentino, G.; Constantin, G. The Blood−Brain Barrier in Alzheimer’s Disease. Neurobiol. Dis. 2017, 107, 41−56. (16) Kuo, Y. C.; Lee, C. L.; Rajesh, R. Regulation of Human Brain Vascular Pericytes and Human Astrocytes in a Blood−Brain Barrier Model Using Human Brain Microvascular Endothelial Cells: Expression of TGF-β1, VEGF, MMP-9 and P-gp. J. Taiwan Inst. Chem. Eng. 2018, 86, 9−17. (17) Jallouli, Y.; Paillard, A.; Chang, J.; Sevin, E.; Betbeder, D. Influence of Surface Charge and Inner Composition of Porous Nanoparticles to Cross Blood−Brain Barrier In Vitro. Int. J. Pharm. 2007, 344, 103−109. (18) Kuo, Y. C.; Hong, T. Y. Delivering Etoposide to the Brain Using Catanionic Solid Lipid Nanoparticles With Surface 5-HTmoduline. Int. J. Pharm. 2014, 465, 132−142. (19) Grimaldi, B.; Fillion, G. 5-HT-moduline Controls Serotonergic Activity: Implication in Neuroimmune Reciprocal Regulation Mechanisms. Prog. Neurobiol. 2000, 60, 1−12. (20) Kubera, M.; Maes, M.; Kenis, G.; Kim, Y.-K.; Lason, W. Effects of Serotonin and Serotonergic Agonists and Antagonists on the Production of Tumor Necrosis Factor α and Interleukin-6. Psychiatry Res. 2005, 134, 251−258. (21) Sabogal-Guaqueta, A. M.; Munoz-Manco, J. I.; Ramirez-Pineda, J. R.; Lamprea-Rodriguez, M.; Osorio, E.; Cardona-Gomez, G. P. The Flavonoid Quercetin Ameliorates Alzheimer’s Disease Pathology and Protects Cognitive and Emotional Function in Aged Triple Transgenic Alzheimer’s Disease Model Mice. Neuropharmacology 2015, 93, 134−145. (22) Kuo, Y. C.; Rajesh, R. Targeted Delivery of Rosmarinic Acid Across the Blood−Brain Barrier for Neuronal Rescue Using Polyacrylamide-Chitosan-Poly(Lactide-co-Glycolide) Nanoparticles with Surface Cross-Reacting Material 197 and Apolipoprotein E. Int. J. Pharm. 2017, 528, 228−241. (23) Heldt, N.; Gauger, M.; Zhao, J.; Slack, G.; Pietryka, J.; Li, Y. Characterization of a Polymer-Stabilized Liposome System. React. Funct. Polym. 2001, 48, 181−191. (24) Walde, P. Surfactant Assemblies and their Various Possible Roles for the Origin(S) of Life. Origins Life Evol. Biospheres 2006, 36, 109−150. (25) Jass, J.; Tjarnhage, T.; Puu, G. From Liposomes to Supported, Planar Bilayer Structures on Hydrophilic and Hydrophobic Surfaces: An Atomic Force Microscopy Study. Biophys. J. 2000, 79, 3153−3163. (26) Gidwani, M.; Singh, A. V. Nanoparticle Enabled Drug Delivery Across the Blood Brain Barrier: In Vivo and In Vitro Models, opportunities and challenges. Curr. Pharm. Biotechnol. 2014, 14, 1201−1212. (27) Roney, C.; Kulkarni, P.; Arora, V.; Antich, P.; Bonte, F.; Wu, A.; Mallikarjuana, N. N.; Manohar, S.; Liang, H.-F.; Kulkarni, A. R.; Sung, H.-W.; Sairam, M.; Aminabhavi, T. M. Targeted Nanoparticles for Drug Delivery Through the Blood−Brain Barrier for Alzheimer’s Disease. J. Controlled Release 2005, 108, 193−214. (28) Callahan, M. K.; Williams, K. A.; Kivisakk, P.; Pearce, D.; Stins, M. F.; Ransohoff, R. M. CXCR3Marks CD4+ Memory T lymphocytes

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], Tel: 886-5-272-0411 ext. 33459, Fax: 886-5-272-1206. ORCID

Yung-Chih Kuo: 0000-0002-5837-8546 Rajendiran Rajesh: 0000-0002-1272-9523 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is supported by the Ministry of Science and Technology of the Republic of China.

■

REFERENCES

(1) Fitzpatrick, A. W. P.; Falcon, B.; He, S.; Murzin, A. G.; Murshudov, G.; Garringer, H. J.; Crowther, R. A.; Ghetti, B.; Goedert, M.; Scheres, S. H. W. Cryo-EM Structures of tau Filaments from Alzheimer’s Disease. Nature 2017, 547, 185−190. (2) Kandimalla, R.; Thirumala, V.; Reddy, P. H. Is Alzheimer’s Disease a Type 3 Diabetes? A Critical Appraisal. Biochim. Biophys. Acta, Mol. Basis Dis. 2017, 1863, 1078−1089. (3) Takahashi, R. H.; Nagao, T.; Gouras, G. K. Plaque Formation and the Intraneuronal Accumulation of β-amyloid in Alzheimer’s Disease. Pathol. Int. 2017, 67, 185−193. (4) Qiang, W.; Yau, W.-M.; Lu, J.-X.; Collinge, J.; Tycko, R. Structural Variation in Amyloid-β Fibrils from Alzheimer’s Disease Clinical Subtypes. Nature 2017, 541, 217−221. (5) Kuo, Y. C.; Rajesh, R. A Critical Overview of Therapeutic Strategy and Advancement for Alzheimer’s Disease Treatment. J. Taiwan Inst. Chem. Eng. 2017, 77, 92−105. (6) Ismaili, L.; Refouvelet, B.; Benchekroun, M.; Brogi, S.; Brindisi, M.; Gemma, S.; Campiani, G.; Filipic, S.; Agbaba, D.; Esteban, G.; Unzeta, M.; Nikolic, K.; Butini, S.; Marco-Contelles, J. Multitarget Compounds Bearing Tacrine- and Donepezil-like Structural and Functional Motifs for the Potential Treatment of Alzheimer’s Disease. Prog. Neurobiol. 2017, 151, 4−34. (7) Graham, W. V.; Bonito-Oliva, A.; Sakmar, T. P. Update on Alzheimer’s Disease Therapy and Prevention Strategies. Annu. Rev. Med. 2017, 68, 413−430. (8) Alzheimer’s Association. 2017 Alzheimer’s disease facts and figures. Alzheimer's Dementia 2017, 13, 325−373. (9) Weber, B.; Kappel, C.; Scherer, M.; Helm, M.; Bros, M.; Grabbe, S.; Barz, M. PeptoSomes for Vaccination: Combining Antigen and Adjuvant in Polypept(o)ide-Based Polymersomes. Macromol. Biosci. 2017, 10, 1700061. (10) Liang, C.; Li, F.; Wang, L.; Zhang, Z.-F.; Wang, C.; He, B.; Li, J.; Chen, Z.; Shaikh, A. B.; Liu, J.; Wu, X.; Peng, S.; Dang, L.; Guo, B.; He, X.; Au, D. W. T.; Lu, C.; Zhu, H.; Zhang, G.; Lu, A.; Zhang, G. Tumor Cell-Targeted Delivery of CRISPR/Cas9 by AptamerFunctionalized Lipopolymer for Therapeutic Genome Editing of VEGFA in Osteosarcoma. Biomaterials 2017, 147, 68−85. I


Article

ACS Biomaterials Science & Engineering that are Competent to Migrate Across a Human Brain Microvascular Endothelial Cell Layer. J. Neuroimmunol. 2004, 153, 150−157. (29) Kuo, Y. C.; Lee, Y. J. Rescuing Cholinergic Neurons from Apoptotic Degeneration by Targeting of Serotonin Modulator-and Apolipoprotein E-conjugated Liposomes to the Hippocampus. Int. J. Nanomed. 2016, 11, 6809−6824. (30) Lu, W.; Tan, Y.-Z.; Hu, K.-L.; Jiang, X.-G. Cationic Albumin Conjugated Pegylated Nanoparticle with its Transcytosis Ability and Little Toxicity Against Blood−Brain Barrier. Int. J. Pharm. 2005, 295, 247−260. (31) Roshanzamir, F.; Yazdanparast, R. Quercetin Attenuates Cell Apoptosis of Oxidant-Stressed SK-N-MC Cells while Suppressing Upregulation of the Defensive Element, HIF-1α. Neuroscience 2014, 277, 780−793. (32) Bana, L.; Minniti, S.; Salvati, E.; Sesana, S.; Zambelli, V.; Cagnotto, A.; Orlando, A.; Cazzaniga, E.; Zwart, R.; Scheper, W.; Masserini, M.; Re, F. Liposomes Bi-functionalized with Phosphatidic Acid and an ApoE-derived Peptide Affect Aβ Aggregation Features and Cross the Blood−Brain-Barrier: Implications for Therapy of Alzheimer Disease. Nanomedicine 2014, 10, 1583−1590. (33) Re, F.; Cambianica, I.; Sesana, S.; Salvati, E.; Cagnotto, A.; Salmona, M.; Couraud, P.-O.; Moghimi, S. M.; Masserini, M.; Sancini, G. Functionalization with ApoE-derived Peptides Enhances the Interaction with Brain Capillary Endothelial Cells of Nanoliposomes Binding Amyloid-beta Peptide. J. Biotechnol. 2011, 156, 341−346. (34) Riedl, S. F.; Shi, Y. Molecular Mechanisms of Caspase Regulation During Apoptosis. Nat. Rev. Mol. Cell Biol. 2004, 5, 897− 907. (35) Nakagawa, S.; Deli, M. A.; Kawaguchi, H.; Shimizudani, T.; Shimono, T.; Kittel, A.; Tanaka, K.; Niwa, M. A New Blood−Brain Barrier Model Using Primary Rat Brain Endothelial Cells, Pericytes and Astrocytes. Neurochem. Int. 2009, 54, 253−263. (36) Armulik, A.; Genove, G.; Mae, M.; Nisancioglu, M. H.; Wallgard, E.; Niaudet, C.; He, L.; Norlin, J.; Lindblom, P.; Strittmatter, K.; Johansson, B. R.; Betsholtz, C. Pericytes Regulate the Blood−Brain Barrier. Nature 2010, 468, 557−561. (37) Sarhan, H.; Grimaldi, B.; Hen, R.; Fillion, G. 5-HT1B Receptors Modulate Release of [3H]dopamine from Rat Striatal Synaptosomes: Further Evidence Using 5-HT Moduline, Polyclonal 5-HT1B Receptor Antibodies and 5-HT1B Receptor Knock-Out Mice. NaunynSchmiedeberg's Arch. Pharmacol. 2000, 361, 12−18. (38) Martorana, A.; Koch, G. Is Dopamine Involved in Alzheimer’s Disease? Front. Aging Neurosci. 2014, 6, 252. (39) Melo, J. B.; Agostinho, P.; Oliveira, C. R. Oliveira, Involvement of Oxidative Stress in the Enhancement of Acetylcholinesterase Activity Induced by Amyloid beta-peptide. Neurosci. Res. 2003, 45, 117−127. (40) Parnetti, L.; Amici, S.; Lanari, A.; Romani, C.; Antognelli, C.; Andreasen, N.; Minthon, L.; Davidsson, P.; Pottel, H.; Blennow, K.; Gallai, V. Cerebrospinal Fluid Levels of Biomarkers and Activity of Acetylcholinesterase (AChE) and Butyrylcholinesterase in AD Patients Before and After Treatment with Different Ache Inhibitors. Neurol. Sci. 2002, 23, s95−s96. (41) Butterfield, D. A.; Lauderback, C. M. Lipid Peroxidation and Protein Oxidation in Alzheimer’s Disease Brain: Potential Causes and Consequences Involving Amyloid β-peptide-Associated Free Radical Oxidative Stress. Free Radical Biol. Med. 2002, 32, 1050−1060. (42) Gunn, A. P.; Wong, B. X.; Johanssen, T.; Griffith, J. C.; Masters, C. L.; Bush, A. I.; Barnham, K. J.; Duce, J. A.; Cherny, R. A. Amyloidβ Peptide Aβ3pE-42 Induces Lipid Peroxidation, Membrane Permeabilization, and Calcium Influx in Neurons. J. Biol. Chem. 2016, 291, 6134−6145.

J


Protection against Neurodegeneration in the Hippocampus Using

Recommend Documents