GM1-Modified Lipoprotein-like Nanoparticle: Multifunctional

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GM1-Modified Lipoprotein-like Nanoparticle: Multifunctional Nanoplatform for the Combination Therapy of Alzheimer's Disease Meng Huang,†,^ Meng Hu,†,^ Qingxiang Song,† Huahua Song,† Jialin Huang,† Xiao Gu,† Xiaolin Wang,† Jun Chen,‡ Ting Kang,‡ Xingye Feng,‡ Di Jiang,‡ Gang Zheng,§ Hongzhuan Chen,*,† and Xiaoling Gao*,† †

Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, People's Republic of China, ‡Department of Pharmaceutics, Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, People's Republic of China, and §Department of Medical Biophysics and Ontario Cancer Institute, University of Toronto, Toronto, Ontario M5G 1L7, Canada. ^M. Huang and M. Hu contributed equally.

ABSTRACT Alzheimer's disease (AD) exerts a heavy health

burden for modern society and has a complicated pathological background. The accumulation of extracellular β-amyloid (Aβ) is crucial in AD pathogenesis, and Aβ-initiated secondary pathological processes could independently lead to neuronal degeneration and pathogenesis in AD. Thus, the development of combination therapeutics that can not only accelerate Aβ clearance but also simultaneously protect neurons or inhibit other subsequent pathological cascade represents a promising strategy for AD intervention. Here, we designed a nanostructure, monosialotetrahexosylganglioside (GM1)-modified reconstituted high density lipoprotein (GM1-rHDL), that possesses antibody-like high binding affinity to Aβ, facilitates Aβ degradation by microglia, and Aβ efflux across the blood
brain barrier (BBB), displays high brain biodistribution efficiency following intranasal administration, and simultaneously allows the efficient loading of a neuroprotective peptide, NAP, as a nanoparticulate drug delivery system for the combination therapy of AD. The resulting multifunctional nanostructure, RNAP-GM1-rHDL, was found to be able to protect neurons from Aβ1
42 oligomer/glutamic acid-induced cell toxicity better than GM1-rHDL in vitro and reduced Aβ deposition, ameliorated neurologic changes, and rescued memory loss more efficiently than both RNAP solution and GM1-rHDL in AD model mice following intranasal administration with no observable cytotoxicity noted. Taken together, this work presents direct experimental evidence of the rational design of a biomimetic nanostructure to serve as a safe and efficient multifunctional nanoplatform for the combination therapy of AD. KEYWORDS: reconstituted high-density lipoprotein . nanoplatform . Alzheimer's disease . amyloid-β . combination therapy . GM1 ganglioside . intranasal administration

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ementia remains one of the biggest global public health challenges. According to the World Alzheimer Report 2014, the number of people living with dementia is around 44 million worldwide and estimated to double in 2030 and triple by 2050. Alzheimer's disease (AD), the most prevalent form of dementia, with relatively long disease course and high cost for medical care, has become a tremendous burden for both families and society. Unfortunately, no disease-modifying treatments are currently available. Therefore, the HUANG ET AL.

development of novel AD therapeutics is urgently needed. AD is caused by multiple pathologies. One distinctive pathological change in the AD brain is the aberrant accumulation of extracellular β-amyloid (Aβ), which is believed to initiate the pathological cascade of tau protein hyperphosphorylation, neurofibrillary tangles, synaptic dysfunction, neuronal death, and eventually loss of cognitive function.1 The imbalance between Aβ production and clearance is suggested to cause its deposition.2 Especially, recent VOL. XXX

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* Address correspondence to [email protected], [email protected]. Received for review May 23, 2015 and accepted October 6, 2015. Published online 10.1021/acsnano.5b03124 C XXXX American Chemical Society

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hold great potential to be developed as a multifunctional nanoplatform for the combination therapy of AD. Neuroprotective peptides have shown great potential for the treatment of AD.28,29 However, their clinical application was largely hindered by their poor stability and inability to cross the BBB. Here, to evaluate the potential of GM1-rHDL as a flexible nanoplatform for the combination therapy of AD, NAP (NAPVSIPQ), a femtomolar-acting neuroprotective peptide that was derived from the activity-dependent neuroprotective protein (ADNP) and has been used to protect neurons from damage induced by N-methyl-D-aspartic acid (NMDA),30 tetrodotoxin,30 and Aβ1
42,31 was selected as a model drug to be loaded into GM1-rHDL. The neuroprotective activity of the NAP-loaded GM1-rHDL (RNAP-GM1-rHDL) against Aβ1
42 oligomer/glutamate acid-induced toxicity was evaluated in vitro. Furthermore, its protective effect on memory loss and neurologic damage in AD model mice following intranasal administration was also investigated.

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studies found that the common late-onset form of AD is characterized by an overall impairment in Aβ clearance but not in Aβ production.3,4 Therefore, immunotherapy that can efficiently accelerate Aβ clearance has been regarded as one of the most promising strategies for AD therapy.5 However, recent clinical trials have witnessed the failure of immunotherapy in improving cognitive function in patients.6
8 Besides the autoimmunity-related adverse effects induced by immunotherapy, the major reason for such failure lies in the lack of function of the immunotherapeutics in inhibiting the pathological cascades induced by Aβ accumulation. It has been widely accepted that the secondary pathological processes such as dysfunction of synapse and neuron loss could independently aggravate the severity of AD, once initiated by Aβ.9 Thus, the development of novel multifunctional therapeutics that can not only accelerate Aβ clearance but also inhibit the subsequent pathological cascades represents a promising strategy for AD therapy.10
12 The nanoparticulate drug delivery system has been widely studied as a powerful platform for combination therapy of complex diseases such as cancer and AIDS.13
15 For the combination treatment of AD, the nanoparticulate drug delivery system that possesses both blood
brain barrier (BBB) permeability and high Aβ-binding affinity is of special interest. Previously, we have demonstrated that the apoE3-reconstituted highdensity lipoprotein (rHDL), a 20 nm nanoparticle, possessed BBB permeability and high Aβ-binding affinity, decreased Aβ deposition, ameliorated neurologic changes, and rescued memory deficits in an AD animal model.16 Considering that rHDL is a versatile class of biologically inspired nanostructures that can also carry both hydrophobic and hydrophilic agents,17 it holds great potential to be developed into a multifunctional nanoplatform for the combination therapy of AD. Previous work showed that high Aβ-binding affinity is indispensable for the development of an antibody for efficient Aβ clearance.18,19 In addition, lipidated apoE, which possessed higher Aβ-binding affinity than apoE alone, also exhibited higher Aβ clearance activity.20 Here, to develop rHDL into a more efficient multifunctional nanoplatform for the combination therapy of AD, we explored whether the Aβ-clearance activity of rHDL could be further enhanced by improving its Aβ-binding affinity. To achieve this goal, monosialotetrahexosylganglioside (GM1), an anionic amphiphilic lipid possessing Aβ-binding ability,21,22 was incorporated into the lipid membrane of rHDL to construct a novel nanostructure, GM1-modified rHDL (GM1-rHDL). Its Aβ-binding affinity and in vivo and in vitro Aβ clearance abilities were evaluated and compared with those of rHDL. As various therapeutics such as siRNA,23 peptide,24,25 and chemicals26,27 can be efficiently loaded into rHDL-like nanoparticles, GM1-rHDL was also expected to be able to carry various cargoes and

RESULTS AND DISCUSSION Design, Preparation, and Characterization of GM1-rHDL and rNAP-GM1-rHDL. rHDL/GM1-rHDL was prepared by incubating lipid-free apoE3 with 1,2-dimyristoyl-sn-glycero3-phosphocholine (DMPC) liposome or GM1-DMPC liposome, respectively, according to our previously reported method.16 1,10 -Dioctadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (DiI) or 3,30 -dioctadecyloxacarbocyanine perchlorate (DiO) (1% to DMPC, w/w) was incorporated into the membrane of rHDL or GM1rHDL for fluorescent labeling.16,32 The narrow size distributions of both rHDL and GM1-rHDL were confirmed by dynamic light scattering (DLS), with an average particle size of 24.64 ( 3.59 and 23.67 ( 6.68 nm, respectively (Figure 1A,B, Table 1). The zeta potential of GM1-rHDL (
14.20 ( 0.66 mV) was more negative than that of rHDL (
8.06 ( 0.78 mV) (Table 1), suggesting the efficient incorporation of GM1, an anionic amphiphilic lipid, into the lipid membrane. Fluorescent and radio labeling did not change either the size or the zeta potential of both rHDL and GM1-rHDL (Table 1). Transmission electron microscopy (TEM) analysis showed that both rHDL and GM1-rHDL generally presented a circular shape with diameters around 20 nm (Figure 1D). Compared with rHDL, GM1-rHDL seemed to exhibit higher dispersity, which was possibly caused by its lower zeta potential, which could prevent the nanostructures from adhesion. Cryoelectron microscopy was used to further characterize the structure of rHDL and GM1-rHDL. As shown in Figure 1E, the random orientation of the nanoparticles in ice resulted in mainly two types of projections for both rHDL and GM1-rHDL: rectangular projections with two parallel bands of high density and circular projections with a ring of high density. Such conformations were similar to those of the apoA I-containing rHDL33,34 and suggested that VOL. XXX

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ARTICLE Figure 1. Characterization of the nanoparticles. Particle size distribution of (A) rHDL, (B) GM1-rHDL, and (C) RNAP-GM1-rHDL analyzed by dynamic light scattering via a Zetasizer. (D) Morphology and particle size of rHDL, GM1-rHDL, RNAP-GM1-DMPC liposome, and RNAP-GM1-rHDL under a transmission electron microscope after negative staining with sodium phosphotungstate solution (2%, w/v). Scale bar: 20 nm. (E) Morphology and particle size of rHDL and GM1-rHDL under cryoelectron microscopy. Rectangular projections with two parallel bands of high density are indicated by arrowheads, and circular projections with a ring of high density are pointed out by arrows. Scale bar: 50 nm.

TABLE 1. Particle Size and Zeta Potential of Nanoparticles

rHDL GM1-rHDL RNAP-GM1-rHDL RNAP-GM1-DMPC liposome GM1-DMPC liposome DiI-rHDL DiI-GM1-rHDL DiO-GM1-rHDL 125 I-rHDL 125 I-GM1-rHDL

PDI

particle size (d, nm)

zeta potential (mV)

0.260 0.239 0.218 0.192 0.202 0.236 0.252 0.265 0.281 0.178

24.64 ( 3.59 23.27 ( 6.68 25.42 ( 1.18 39.36 ( 0.92 55.17 ( 5.11 22.09 ( 2.64 21.38 ( 3.13 23.65 ( 2.29 23.09 ( 5.73 22.14 ( 4.57


8.06 ( 0.78
14.20 ( 0.66
15.70 ( 0.93
13.30 ( 0.64
21.70 ( 0.15
9.59 ( 0.19
19.50 ( 0.50
15.80 ( 1.16
9.02 ( 0.57
15.10 ( 0.98

GM1-rHDL had a discoidal shape and shared similar structural features with natural nascent HDL and those rHDL containing N-terminal apoE3, apoA I, or apoA I mimetic peptide.35
38 As the lipidization process and the formation of a discoidal structure are important and essential in improving the affinity of apoE to both its receptors and Aβ,20,39,40 GM1-rHDL was also HUANG ET AL.

expected to possess high binding affinity to both apoE receptors and Aβ. To verify the potential of GM1-rHDL as a flexible nanoplatform for the combination therapy of AD, a neuroprotective octapeptide, NAP, was chosen as a model drug and loaded into GM1-rHDL. In order to load NAP into GM1-rHDL, a hybrid peptide containing a NAP, an R-helix sequence (Ac-FAEKFKEAVKDYFAKFWD), and a GSG spacer was synthesized. The R-helix sequence exhibits an amphipathic helix structure41 and strong lipid-association properties, which mimic apolipoproteins.23 Before loading RNAP into GM1-rHDL, the lipid-association property of RNAP was confirmed by incubating RNAP with DMPC liposomes at a ratio of 1:20 (RNAP:DMPC, w/w). The size of the obtained nanoparticles was 25.46 ( 2.04 nm, much smaller than that of the DMPC liposome (39.81 ( 1.09 nm), indicating that RNAP perfectly retained its lipid-association property. RNAP-GM1-rHDL was then prepared via a two-step incubation process: first, GM1-DMPC liposomes were incubated with RNAP at a ratio of 1:125 VOL. XXX

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ARTICLE Figure 2. ApoE-concentration-dependent binding of rHDL and GM1-rHDL to Aβ1
42 monomers and oligomers. (A and C) Binding of rHDL and GM1-rHDL to Aβ1
42 monomers, respectively. (B and D) Binding of rHDL and GM1-rHDL to Aβ1
42 oligomers, respectively. The binding affinity was evaluated by surface plasmon resonance analysis, and the kinetic constants of binding were obtained using a 1:1 Langmuir binding model via the BIAevaluation software.

(RNAP:lipid, w/w) overnight to form RNAP-loaded GM1-DMPC liposomes (RNAP-GM1-DMPC liposomes), which were subsequently incubated with apoE3 at a ratio of apoE3:lipid 1:5 (w/w) for 24 h to form RNAPGM1-rHDL. The concentration of RNAP was quantitated by high-performance liquid chromatography (HPLC), and the entrapping efficiency (EE) of RNAP in RNAP-GM1-rHDL was 64.39 ( 12.84%. The particle size of the RNAP-GM1-DMPC liposome (39.36 ( 0.92 nm) was smaller than that of the GM1-DMPC liposome (55.17 ( 5.11 nm), while the size of RNAP-GM1-rHDL (25.42 ( 1.18 nm) was the smallest (Table 1, Figure 1C). The decrease of particle size indicated that both RNAP and apoE3 had been successfully assembled into the lipid nanoparticles following the two-step incubation process. The successful assembly was further confirmed under TEM (Figure 1D), where RNAP-GM1-rHDL exhibited a similar size, morphology, and distribution profile to GM1-rHDL (Figure 1D). Furthermore, the zeta potential of RNAP-GM1-rHDL was similar to that of GM1-rHDL as well (Table 1). GM1-rHDL Binds to Aβ1
42 Monomer and Oligomer with High Affinity. To effectively target Aβ and decrease its extracellular accumulation, agents should possess anti-Aβ antibody-like high Aβ-binding affinity.18,42,43 Aβ1
40 and Aβ1
42 are the most common sequential proteolytic products of amyloid β-protein precursor (APP), while Aβ1
42 is more toxic than Aβ1
40 due to its greater propensity to aggregate.44 Especially, the soluble forms of Aβ, both monomer and oligomer, which have been suggested to be the major toxic forms involved in AD pathology,45 were used to characterize HUANG ET AL.

the Aβ-binding affinity of GM1-rHDL via a surface plasmon resonance (SPR) analysis. For the determination, Aβ1
42 monomer and oligomer were separately immobilized onto two separate flow cells of a CM5 chip via an amino coupling reaction. The reference flow cells were blocked with ethanolamine immediately after activation. An apoE-concentration-dependent binding manner was recorded following the application of GM1-rHDL. The rates of complex aggregation and dissociation were kinetically analyzed using a 1:1 Langmuir binding model to obtain the values of the binding affinity constant (KD). The KD values of GM1rHDL to Aβ1
42 monomer and oligomer were in the subnanomolar range, 2.13  10
10 and 1.51  10
10 M, respectively, which were 46 and 62 times smaller than that of rHDL (Figure 2, Table 2), suggesting that GM1rHDL did exhibit much higher Aβ-binding affinity than rHDL. In contrast, apoE3 alone exhibited much lower binding affinity to Aβ1
42 monomer (KD 2.95  10
8 M) and hardly bound to Aβ1
42 oligomer. In the case of the apoE-free GM1-DMPC liposome, a much lower binding affinity was also observed to both Aβ1
42 monomer and oligomer (KD values of 1.50  10
8 and 4.04  10
8 M, respectively). (The KD value of the GM1-DMPC liposome to Aβ1
42 was calculated on the basis of the same lipid concentration with GM1-rHDL.) In addition, incorporation of other biocompatible lipids such as cardiolipin and sulfatide into the lipid membrane of rHDL failed to enhance the Aβ-binding affinity of rHDL. (The KD values of sulfatide-modified rHDL to Aβ1
42 monomer and Aβ1
42 oligomer were 6.83  10
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Aβ1
42 monomer kass (M
1 s
1)

rHDL GM1-rHDL a

kdiss (s
1)

(1.26 ( 0.61)  10 (8.47 ( 5.54)  105 5

Aβ1
42 oligomer kass (M
1 s
1)

KD (M)
3

(1.17 ( 0.25)  10 (1.41 ( 1.31)  10
4


9

(9.97 ( 2.81)  10 (2.13 ( 1.52)  10
10

kdiss (s
1)

KD (M)
4

(9.30 ( 3.66)  10 (6.58 ( 4.98)  105

(8.00 ( 0.66)  10 (9.93 ( 7.30)  10
5

4

(9.47 ( 4.37)  10
9 (1.51 ( 0.02)  10
10

kass, association rate constant; kdiss, dissociation rate constant; and KD, binding affinity constant.

of cardiolipin-modified rHDL to Aβ1
42 monomer and Aβ1
42 oligomer were 1.50  10
8 and 6.06  10
9 M, respectively.) This evidence suggested that the high Aβ-binding affinity of GM1-rHDL was achieved by the specific synergetic effect of DMPC, GM1, and apoE and also verified our hypothesis that the Aβ-binding affinity of rHDL could be elevated by incorporating a component that also possessed Aβ-binding affinity into the lipid membrane. Such high Aβ-binding affinity of GM1-rHDL was comparable with that of highly efficient anti-Aβ antibody.18 As all the components in GM1-rHDL were biocompatible, GM1-rHDL was expected to exhibit high Aβ clearance activity but avoid the antibodyinduced autoimmunity-related adverse effects. GM1-rHDL Displays Higher Brain Distribution Than rHDL Following Intranasal Administration. Accessing the brain is one of the major prerequisites for agents to exert their therapeutic effects in the central nervous system (CNS). For the treatment of neurodegenerative diseases such as AD, in which long-term intervention is needed, patient compliance is extremely important, and thus noninvasive administration routes are of particular interest. Intranasal delivery, a noninvasive method for brain drug delivery, which can bypass the BBB to allow therapeutics such as peptides, proteins, oligonucleotides, viral vectors, nanocarriers, and even stem cells direct access to the CNS,46,47 seems to be a promising modality. In order to evaluate the brain transport efficiency of GM1-rHDL following intranasal administration, GM1rHDL and rHDL were separately 125I labeled using the Bolton
Hunter procedure as described previously,48 and their radioactivities in different brain sections and in the peripheral organs after nasal dosing were determined with a γ-counter and expressed as percentage of the injected dose per gram of tissue (% ID/g). As shown in Table 3, the Cmax of 125I-rHDL and 125I-GM1rHDL in the cortex þ hippocampus after intranasal administration were 0.0248% and 0.0434% ID/g, respectively. The AUCall values of GM1-rHDL in the cortex þ hippocampus were 0.85-fold higher than that of rHDL, while the AUCall ratios of cortex þ hippocampus to blood (AUCCortexþHippocampus/AUCblood) were comparable between GM1-rHDL and rHDL, suggesting that GM1-rHDL exhibited higher intranasal absorption efficiency than rHDL (Table 3). Similarly, the radioactivities of GM1-rHDL detected in the major organs and the HUANG ET AL.

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a

TABLE 2. Average Affinity Constants Obtained from the Interaction between rHDL/GM1-rHDL and Aβ (n = 3)

125 I-rHDL and I-GM1-rHDL Following Intranasal Administration

TABLE 3. Pharmacokinetic Parameters of 125

125

125

I-rHDL

cortex þ hippocampus

Tmax (h) Cmax (% ID/g) AUCall (% ID/g 3 h) AUCCortexþHippocampus/AUCblood

I-GM1-rHDL

cortex þ blood

hippocampus

4 4 0.0248 0.8074 0.1596 5.4945 0.0291

blood

2 4 0.0434 1.333 0.2956 9.8650 0.0300

blood circulation after intranasal administration were also higher than that of rHDL (SI Figure 1). As no evidence of the expression of the receptor for GM1 was found in the nasal cavity, we believed that the higher dispersity of GM1-rHDL over rHDL could be the major reason that facilitated its absorption in the nasal mucosa and distribution into the brain. Such higher dispersity of GM1-rHDL was due to its lower zeta potential, which could prevent the nanostructures from adhesion. For nasal delivery, in which the administration volume is extremely small and the concentrations of the formulations are relatively high, higher dispersity of the formulation is extremely important for higher nasal absorption. It has been suggested that intranasally applied drugs were transported into the brain mainly via both the olfactory and the trigeminal nerve pathways.46,47 Transport via the olfactory pathway accelerates drug delivery to the rostral brain areas, while transport via the trigeminal nerve pathway facilitates direct drug delivery to the caudal brain regions. As shown in Figure 3, 0.167 h after dosing, the radioactivities of both rHDL and GM1-rHDL were detected highest in the olfactory bulb and the brainstem. In addition, the concentration of both rHDL and GM1-rHDL gradually increased from the cortex to the brainstem at most of the time points. This evidence suggested that, following intranasal administration, both rHDL and GM1-rHDL might be transported into the CNS via both the olfactory and the trigeminal nerve pathways. GM1-rHDL Accelerates Microglia-Mediated Degradation and Brain-to-Blood Efflux of Aβ1
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Figure 3. Brain distribution of (A) 125I-rHDL and (B) administration at a lipid dose of 5 mg/kg.

125

I-GM1-rHDL at 0.167, 0.5, 1, 2, 4, 8, and 12 h after intranasal

Figure 4. Effects of rHDL and GM1-rHDL on the cellular uptake, intracellular distribution, and degradation of Aβ1
42 in microglia. (A) Cellular uptake of 2 μg/mL FAM-Aβ1
42 in primary microglia in the presence of rHDL or GM1-rHDL (0, 0.01, 0.05, 0.5, 2 μg/mL) after incubation for 4 h (n = 3). (B) Intracellular Aβ1
42 levels were quantified by ELISA and normalized to total protein after 4 h of co-incubation of Aβ1
42 (2 μg/mL) with rHDL or GM1-rHDL at a total lipid concentration of 0, 1, 10, and 100 μg/mL (n = 3). (C) Pearson's correlation coefficient of 2 μg/mL FAM-Aβ1
42 with DiI-rHDL or DiI-GM1-rHDL at a lipid concentration of 10 μg/mL after 4 h of co-incubation (n = 5). (D) Colocalization of FAM-Aβ1
42 with DiI-rHDL or DiI-GM1-rHDL after 4 h of co-incubation. Arrow: FAM-Aβ1
42 colocalized with GM1-rHDL. Scale bar: 20 μm. *p < 0.05, **p < 0.01, ****p < 0.0001, significantly different.

cerebrospinal fluid (CSF).49 Agents possessing Aβbinding affinity have been shown to accelerate both cell-based Aβ proteolytic digestion and Aβ efflux across the BBB.50 GM1-rHDL, which exhibited high Aβ-binding affinity, was expected to efficiently capture Aβ in the ISF and accelerate Aβ clearance via both of the above mechanisms. Microglia, mononuclear phagocytes that execute microenvironment surveillance in the brain, have been suggested to be the most important cell type mediating HUANG ET AL.

intracerebral cellular Aβ clearance.51 Here, we assumed that GM1-rHDL, which exhibited much higher Aβbinding affinity, would more efficiently capture Aβ and accelerate microglia-mediated Aβ degradation than rHDL. To justify this hypothesis, primary microglia were incubated with Aβ1
42 in the presence of rHDL or GM1-rHDL with the cellular uptake, intracellular localization, and degradation of Aβ1
42 determined. High content screening (HCS), a cell-based quantitative fluorescence imaging system, was applied to measure VOL. XXX

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the cellular uptake level of Aβ1
42 and the colocalization correlation coefficient between Aβ1
42 and rHDL or GM1-rHDL. As shown in Figure 4A, the cellular uptake of Aβ1
42 in primary microglia was facilitated following 4 h co-incubation with GM1-rHDL, with the concentration of GM1-rHDL raised from 0.05 μg/mL to 2 μg/mL, and the cellular uptake of Aβ1
42 increased from 63.3 ( 2.6 to 83.2 ( 2.4 in terms of fluorescence intensity. In contrast, co-incubation with rHDL at the lipid concentration from 0.01 to 2 μg/mL only slightly increased the uptake of Aβ1
42 (Figure 4A). Interestingly, even with a higher amount of Aβ1
42 internalized into the microglia after 4 h of incubation, the intracellular level of Aβ1
42 detected by ELISA in those Aβ1
42 þ GM1-rHDL-treated cells was significantly lower than that of the Aβ1
42 alone-treated controls (Figure 4B). In the rHDL-treated cells, the level of undegraded Aβ1
42 detected by ELISA increased by 52.3% as the concentration of rHDL increased from 1 to 100 μg/mL; in contrast, in the GM1-rHDL-treated cells, the level of undegraded Aβ1
42 decreased by 24.2% as the concentration of GM1-rHDL increased from 1 to 100 μg/mL, suggesting that GM1-rHDL possessed a wider effective working concentration window in accelerating Aβ1
42 degradation than rHDL. The positive Pearson's correlation coefficient (PCC) values of both rHDL and GM1-rHDL to Aβ1
42 suggested that some extent of colocalization existed between Aβ1
42 and rHDL/GM1-rHDL (Figure 4C).52 The PCC of GM1rHDL to Aβ1
42 was 7-fold higher than that of rHDL, indicating that more Aβ1
42 internalized within the cells were colocalized with GM1-rHDL, which may be caused by the higher Aβ1
42-binding affinity of GM1rHDL (Figure 4C,D). This in vitro evidence collectively indicated that the cellular uptake and intracellular degradation of Aβ1
42 in microglia were facilitated in the presence of GM1-rHDL. In addition, colocalization analysis showed that the PCC of DiO-GM1-rHDL to lysosomes in microglia was 0.86 ( 0.01 (SI Figure 2). This generally high value indicated that, following the entrance into the cell, most GM1-rHDL was transported to the lysosome for degradation. The effect of GM1-rHDL in facilitating Aβ1
42 clearance was also evaluated in vivo, in which Aβ1
42 oligomer was 125I labeled (125I-Aβ1
42) and injected into the unilateral hippocampus of ICR mice with GM1-rHDL or rHDL intranasally administrated 30 min ahead. Those animals intrahippocampally injected with 125I-Aβ1
42 and intranasally administrated with PBS were used as the negative control. Ten and 30 min after the Aβ1
42 injection, the animals were sacrificed, and the brains were harvested and subjected to radioactivity measurement. Trichloroacetic acid (TCA) precipitation was performed to detect the intact form of Aβ1
42, the degraded Aβ was indicated by the TCA-unprecipitated 125I radioactivity,53 and Aβ degradation was expressed as the ratio of TCA-unprecipitated 125I

Figure 5. Effects of rHDL and GM1-rHDL on the brain clearance of Aβ1
42 oligomer. Mice were intranasally administrated with rHDL or GM1-rHDL (at the lipid dose of 5 mg/kg) 30 min before intrahippocampally microinjected with 125I-Aβ1
42 oligomer and [14C]inulin (n = 3
5). Mice intranasally administrated with PBS were used as control. (A) Ratio of degraded 125I-Aβ1
42 oligomer to total 125I injected. (B) BEI of 125I-Aβ1
42 oligomer at 10 and 30 min after microinjection. Data represent the mean ( SEM; *p < 0.05, **p < 0.01, significantly different.

radioactivity to that of the total injected radioactivity. Ten minutes after the 125I-Aβ1
42 oligomer injection, the extent of 125I-Aβ1
42 oligomer degradation following the GM1-rHDL treatment was 66% higher than that of the PBS-treated control. In contrast, the extent of 125I-Aβ1
42 oligomer degradation following the rHDL treatment was only 26.8% higher than that of the PBS-treated control (Figure 5A). Combined with the in vitro cellular degradation data, we believed that the facilitated degradation of 125I-Aβ1
42 oligomer could result from the enhanced cellular uptake and degradation of 125I-Aβ1
42 oligomer in the presence of GM1-rHDL. Agents possessing Aβ-binding affinity have also been shown to accelerate Aβ efflux across the BBB.50 Here, the rate of BBB efflux of Aβ1
42 oligomer in the presence of GM1-rHDL was also evaluated via the intracerebral microinjection technique described previously54,55 and expressed as brain efflux index (BEI).56,57 [14C]Inulin, which does not pass across the BBB, was injected simultaneously with 125I-Aβ1
42 oligomer to serve as a reference. As shown in Figure 5B, 10 min after the intrahippocampal Aβ1
42 oligomer injection, the BEI of 125I-Aβ1
42 oligomer was almost the same following the treatment with PBS, rHDL, or GM1-rHDL. But 20 min later, compared with the PBS-treated control, the GM1-rHDL treatment VOL. XXX

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ARTICLE Figure 6. Neuroprotective effects of RNAP-GM1-rHDL on Aβ1
42 oligomer-induced neuronal toxicity. Primary cultured neurons were incubated with drug-free medium (control), 10 μM Aβ1
42 oligomers, and 10 μM Aβ1
42 oligomers in the presence of 10
14 M RNAP, GM1-rHDL (at the same lipid concentration as RNAP-GM1-rHDL), or 10
14 M RNAP-GM1-rHDL, respectively, for 48 h, with (A) the neuronal cell viability, (B) mean neurite length, and (C) mean branch point counts quantified via the Neuronal Profiling Bioapplication software. (D) Morphology of the primary neurons was imaged with HCS. Scale bar: 50 μm. Data represent the mean ( SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, significantly different.

significantly increased the BEI of 125I-Aβ1
42 oligomer by 32.4% (p < 0.05), while the rHDL treatment increased the BEI of 125I-Aβ1
42 oligomer by only 18.8%. Considering the higher affinity between GM1rHDL and 125I-Aβ1
42 than rHDL and 125I-Aβ1
42, these results suggested that besides accelerating the intracerebral cellular degradation of 125I-Aβ1
42 oligomer, GM1-rHDL could also enhance the brain-to-blood efflux of 125I-Aβ1
42 oligomer. rNAP-GM1-rHDL Protects Neurons from Aβ1
42 Oligomer/ Glutamate Acid-Induced Toxicity. The destruction of synapses and neuron death will eventually lead to memory loss and cognitive deficits, and therefore neuroprotection was regarded as a key strategy for AD therapy. Here to evaluate the potential of GM1rHDL as a flexible nanoplatform for the combination therapy of AD, NAP, a neuroprotective peptide, was selected as a model drug to be loaded into GM1-rHDL, and the neuroprotective activity of the obtained nanoformulation against Aβ1
42 oligomer/glutamate HUANG ET AL.

acid-induced toxicity was evaluated. In most cases, synaptic damage emerges long before neuronal death in early AD;58
60 therefore neurite outgrowth was also used here as a key indicator of neuronal function.61 Aβ1
42 oligomers induced morphological neurodegeneration of dystrophic neurites, dendritic simplification, and dendritic spine loss in vitro in cultured neurons and in vivo in adult mouse brain62 and in the surroundings of Aβ deposits post-mortem.63 Thus, the neuronal protection effect of RNAP-GM1-rHDL was evaluated in Aβ1
42 oligomer-induced neuron toxicity. To be efficiently loaded into GM1-rHDL, NAP was fused with an R-helix sequence and the neuroprotective effect of the fusion peptide was verified. For the analysis, NAP and RNAP were separately co-incubated with 10 μM Aβ1
42 oligomer-treated primary neurons for 48 h, at concentrations of both 10
14 and 10
10 M. Exposure to Aβ1
42 oligomers for 48 h significantly reduced the cell viability as well as the neurite length and the branch point counts of neuronal cells (SI Figure 3). VOL. XXX

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Both 10
10 M NAP and 10
10 M RNAP significantly reversed such Aβ1
42 oligomer-induced neuronal toxicity, suggesting that the neuroprotective activity of NAP remained unaffected following its conjugation with the R-helix peptide. Then the neuroprotective effect of RNAP-GM1-rHDL was evaluated and compared with that of RNAP and GM1-rHDL, respectively. As shown in Figure 6A and D, after co-incubation with 10 μM Aβ1
42 oligomers for 48 h at the RNAP concentration of 10
14 M (GM1-rHDL was diluted to the same lipid concentration as that of RNAP-GM1-rHDL), compared with that of the cells treated with Aβ1
42 alone, the viability of those neuronal cells increased by 219%, 180%, and 213% following the treatment with RNAP, GM1-rHDL, and RNAP-GM1-rHDL, respectively (all p < 0.01). Both RNAP and RNAP-GM1-rHDL remarkably reversed Aβ1
42 oligomer-induced neuronal dysfunction by increasing the neurite length (Figure 6B) and branch point counts (Figure 6C). In contrast, GM1-rHDL only increased the viability of Aβ1
42 oligomer-treated neuronal cells but failed to prevent the synaptic dysfunction. These results suggested that RNAP retained its neuroprotective activity after being loaded into GM1-rHDL. The neuroprotective effects of RNAP-GM1-rHDL were further evaluated against glutamic acid-induced neuronal toxicity. Glutamate is one of the most prominent neurotransmitters and plays an important role in neuronal excitation. Excessive glutamate stimulation led to aberration in excitation and then triggered synaptic dysfunction and, eventually, cell death.64
66 Aβ was reported to induce glutamate release in astrocytes and mediate synaptic loss,67 which indicated that glutamate might lead to the cascade triggered by Aβ in synaptic damage and consequent cognitive decline in AD. Here we found that the incubation with 200 μM glutamic acid for 48 h significantly decreased the neurite length and branch point counts of neuronal cells without reducing their viability (Figure 7A). GM1rHDL partly reversed the above glutamic acid-induced neuronal toxicity by increasing the neurite length (Figure 7B) and branch point counts (Figure 7C) by 17.5% and 13.3%, respectively. Such an effect could benefit from the neuroprotective activity of GM1.68 In contrast, RNAP-GM1-rHDL (containing 10
14 M RNAP) exhibited more significant neuroprotection by increasing the neurite length (Figure 7B) and branch point counts (Figure 7C) by 30.0% (p < 0.05) and 27.0% (p < 0.05), respectively. rNAP-GM1-rHDL Reduces Aβ Deposition, Ameliorates Neurologic Damage, and Rescues Memory Deficits in AD Model Mice. Accumulating evidence showed that the coadministration of Aβ1
42 with ibotenic acid (IBO), a neurotoxicant to hippocampal cholinergic neurons,69 produced drastic neuronal loss, induced working memory deficits,70
73 and provided a useful model for studying the pathogenetic mechanisms involved in AD.74,75

Figure 7. Neuroprotective effects of RNAP-GM1-rHDL on glutamic acid-induced neuronal toxicity. Primary cultured neurons were incubated with drug-free medium (control), 200 μM glutamic acid, and 200 μM glutamic acid in the presence of 10
14 M RNAP, GM1-rHDL (at the same lipid concentration as RNAP-GM1-rHDL), or 10
14 M RNAP-GM1rHDL, respectively, for 48 h, with (A) the neuronal cell viability, (B) mean neurite length, and (C) mean branch point counts quantified via the Neuronal Profiling Bioapplication software. Data represent the mean ( SEM; *p < 0.05, **p < 0.01, ***p < 0.001, significantly different.

Here, to evaluate the in vivo neuroprotective effects of RNAP-GM1-rHDL, ICR mice were intrahippocampally co-injected with Aβ1
42 and IBO to serve as the AD animal model with mice injected with artificial CSF applied as the normal control (sham). Two days after the intrahippocampal injection, the model mice were daily intranasally administrated for 2 weeks with RNAP, rHDL, GM1-rHDL, and RNAP-GM1-rHDL, respectively, with those animals intranasally dosed with PBS as the negative control (AD). Subsequently, the spatial learning and memory of the animals were assessed via the Morris water maze (MWM) test. As shown in Figure 8, compared with the sham control, the AD model mice showed deficits in their learning performance in terms of escape latency (Figure 8A) and time spent in a targeted quadrant after removing the platform (Figure 8B), VOL. XXX

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ARTICLE Figure 8. RNAP-GM1-rHDL rescued memory deficits in AD model mice. AD model mice were daily intranasally administrated with rHDL, GM1-rHDL, and RNAP-GM1-rHDL at the lipid dose of 5 mg/kg for 2 weeks. The dose for the RNAP solution group was 24 μg/kg. As negative and normal controls, AD model mice and sham control mice were nasally treated with 10 μL of PBS daily for 2 weeks, respectively. (A) Escape latency; (B) percentage of time spent in the quadrant where the escape platform was placed before; (C) representative swimming path. Data represent the mean ( SEM; *p < 0.05, **p < 0.01, ***p < 0.001 significantly different from AD model mice treated with PBS; #p < 0.05, significantly different from AD model mice treated with RNAP solution.

Figure 9. Morphological evaluation of the in vivo neuroprotective effects of RNAP-GM1-rHDL. Nissl staining of neurons in (A) the dentate gyrus region, (B) the CA1 region of the hippocampus, and (C) the cortex of the AD model mice daily treated with PBS, RNAP solution, rHDL, GM1-rHDL, or RNAP-GM1-rHDL for 2 weeks. Neuron shrinkage is indicated by arrowheads. Scale bar: 50 μm.

indicating that the intrahippocampal co-injection of Aβ1
42 and IBO did induce learning and memory dysfunction in ICR mice. During the 5-day training process, those animals treated with RNAP solution, rHDL, or GM1-rHDL showed slightly improved spatial learning and memory in terms of escape latency, while those administered RNAP-GM1-rHDL exhibited significantly shorter latency than PBS-treated AD model mice (Figure 8A). Similarly, in the probe trail where the platform was removed, compared with the rHDLand GM1-rHDL-treated animals, the RNAP-GM1-rHDLtreated ones spent the longest swimming time in a targeted platform quadrant, suggesting that remarkable improvement in searching strategy was achieved HUANG ET AL.

following the intranasal administration of RNAP-GM1rHDL (Figure 8B,C). Moreover, the neuroprotective effects of RNAPGM1-rHDL were evaluated morphologically following Nissl and HE staining. Compared with the sham control mice, neuron shrinkage was observed in both the hippocampus (Figure 9A,B, Figure 10) and the cortex (Figure 9C) of the PBS-treated AD model mice. In contrast, RNAP-GM1-rHDL treatment significantly ameliorated the impairment of neuronal integrity, while RNAP and GM1-rHDL treatment did not show such obvious amelioration. Immunohistochemical analysis was also performed to evaluate the ability of RNAPGM1-rHDL in reducing Aβ deposition. It was found that VOL. XXX

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ARTICLE Figure 10. Morphological evaluation of the in vivo neuroprotective effects of RNAP-GM1-rHDL. HE staining of neurons in the dentate gyrus region of AD model mice that were daily treated with PBS, RNAP solution, rHDL, GM1-rHDL, or RNAP-GM1-rHDL for 2 weeks. Neuron shrinkage is indicated by arrowheads. Scale bar: 50 μm.

Figure 11. RNAP-GM1-rHDL reduced Aβ deposition (brown signals as indicated by arrowheads) in the hippocampus of AD model mice. AD model mice were daily intranasally administrated with rHDL, GM1-rHDL, and RNAP-GM1-rHDL at the lipid dose of 5 mg/kg for 2 weeks. The dose for the RNAP solution group was 24 μg/kg. As negative and normal controls, AD model mice and sham control mice were nasally treated with 10 μL of PBS daily for 2 weeks, respectively. The brain sections were immunostained with anti-Aβ antibody 6E10. Scale bar: 50 μm.

RNAP-GM1-rHDL decreased Aβ deposition in the way comparable with GM1-rHDL, but more efficiently than rHDL (Figure 11). Taken together, two-week daily treatment with RNAP-GM1-rHDL effectively reduced Aβ deposition, ameliorated neurologic damage, and rescued memory deficits in mice intrahippocampally co-injected with Aβ1
42 and IBO. Finally, the animals were sacrificed, with the major organs collected for HE staining to evaluate the biosafety of RNAP-GM1-rHDL for AD therapy. No obvious morphological alterations were microscopically observed in the hearts, livers, spleens, or kidneys of the RNAP-GM1-rHDL-treated mice (SI Figure 4). The nasal mucosal toxicity of RNAP-GM1-rHDL was also assessed HUANG ET AL.

by detecting the immunoreactivity of neuron-specific enolase (NSE), the marker of olfactory receptor neurons,76
78 in the nasal mucosa. No differences were observed among the mice administered RNAP solution, GM1-rHDL, RNAP-GM1-rHDL, and PBS, indicating the biosafety of these nanoparticles on the olfactory nerves (SI Figure 5). These data suggested that, at least partially if not all, the in vivo administration of RNAPGM1-rHDL was safe under the current dosing regimen. CONCLUSIONS In summary, GM1-rHDL, a biologically inspired nanoparticle, was constructed here as a multifunctional nanoplatform for the combination therapy of AD. With VOL. XXX

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protected neurons against Aβ1
42 oligomer/glutamic acid-induced cell toxicity at a femtomolar concentration of RNAP; moreover, two-week daily intranasal administration of RNAP-GM1-rHDL significantly reduced Aβ deposition, ameliorated neurologic changes, and rescued the memory loss in an AD model mice without inducing visible toxicity. Taken together, our work here presented the therapeutic potential of GM1-rHDL, not only in accelerating Aβ clearance but also in inhibiting the subsequent pathological cascades, as a promising multifunctional nanoplatform for the combination therapy of AD.

MATERIALS AND METHODS

was removed via an Amicon Ultra-4 centrifugal filter (Merck Millipore) of 30 kDa weight cutoff following the manufacturer's instruction. The particle size distributions and zeta potential of all the nanoformulations were measured via a Zetasizer Nano-ZS90 system (Malvern Instruments, U.K.) with a 4.0 mW He
Ne laser at 633 nm and a detector angle of 90. The morphology and size of GM1-rHDL and RNAP-GM1-rHDL were observed under a Hitachi H-7650 transmission electron microscope (Hitachi, Inc., Japan) after negative staining with a 2% sodium phosphotungstate solution. For cryoelectron microscopy analysis, samples were prepared using a FEI Vitrobot (FEI, Holland). The frozen hydrated samples were analyzed at 200 kV at
180 C, using a FEI Tecnai F20 electron microscope (FEI, Holland). Encapsulation Efficiency of rNAP-GM1-rHDL. For the analysis, RNAP-GM1-rHDL was first dissolved in methanol to release RNAP. After centrifugation at 12 000 rpm for 10 min, the concentration of RNAP in the supernatant was measured via an HPLC system (Shimadzu LC-20A system, Japan). Separation was performed via a C18 reverse-phase column (5 μm particle size, 150 mm  4.6 mm i.d., YMC, Japan) with the mobile phase composed of 0.1% (v/v) trifluoroacetic acid in acetonitrile/0.1% trifluoroacetic acid in water (37.5:62.5) at a flow rate of 1.2 mL/min and column temperature at 40 C. The detection wavelength was set at 215 nm. The retention time of RNAP was about 8.5 min. The calibration curve was linear in the range 20
500 μg/mL with an R square of 0.999. EE was calculated by dividing the amount of RNAP detected by the theoretic amount of RNAP added. Preparation of Aβ1
42 Monomer and Oligomer. Aβ1
42 was dissolved in hexafluoroisopropanol (HFIP) at 1 mg/mL and stored at
20 C. Before use, HFIP was allowed to evaporate, and the peptide was resuspended in dimethyl sulfoxide (DMSO) at a concentration of 5 mM and bath sonicated for 10 min to obtain the monomeric preparation. For the preparation of Aβ1
42 oligomer, monomeric peptide DMSO solution (5 mM) was diluted to 100 μM in deionized water and incubated at 4 C for 24 h. The presence of oligomer in these preparations has been previously confirmed and characterized.79 SPR Analysis. SPR analysis was performed on a Biacore T200 instrument (GE Healthcare, USA). Aβ1
42 monomer and oligomer were immobilized as previously described.16 The parallel flow cell activated with EDC/NHS and then blocked with 1 M ethanolamine was used as the reference channel. Experiments were conducted with PBS (pH 7.4) as the running buffer, and the analyte was injected at a flow rate of 30 μL/min. Series concentration of GM1-rHDL/rHDL (diluted in 0.01 M PBS, pH 7.4, containing 0
200 nM apoE3) was injected into the flow system, and the kinetic constants of binding were obtained using a 1:1 Langmuir binding model via BIAevaluation software. Quantification of Cellular Uptake of Aβ. Primary microglia were plated at a density of 1 104 cells/well in a 96-well plate in DMEM containing 10% FBS and cultured for 24 h to allow cell attachment. The cells were then incubated with FAM-Aβ1
42 (2 μg/mL) in serum-free DMEM in the presence of rHDL or

Materials. DMPC and GM1 were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Full-length apoE3 was provided by Peprotech (Rocky Hill, NJ, USA). Aβ1
42, DiI, DiO, and Aβ ELISA kits were purchased from Invitrogen (Carlsbad, CA, USA). FAM-Aβ1
42 was provided by AnaSpec (Fremont, CA, USA). NAP (NAPVSIPQ) and RNAP (AC-FAEKFKEAVKDYFAKFWD-GSGNAPVSIPQ) peptide were synthesized by the GL Biochem (Shanghai, China). All cell culture reagents were purchased from Gibco (Grand Island, NY, USA) unless otherwise indicated. Cells. Primary microglial cells were derived from the cortex of Sprague
Dawley (SD) rats at postnatal day 1
2 as previously described.16 Cells were maintained in DMEM containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). Primary neurons were derived from the cortex of embryonic day 13
15 (E13
15) SD rat embryos as previously described with minor modifications.61 Briefly, the embryos were collected and the cortices were dissected in cold DMEM. The obtained cortical tissues were pooled together and transferred to HBSS containing 0.8 mg/mL papain (Worthington Biochemical, Lakewood, NJ, USA) and 0.24 mg/mL L-cysteine (Sigma-Aldrich, St. Louis, MO, USA) and then incubated for 5 min at 37 C. The enzymatic dissociation was ended following the addition of DMEM containing 10% FBS (v/v), 2 mM L-glutamine, 1% penicillin/streptomycin, and 2000 IU/ml DNase. After centrifugation at 800g for 5 min, the pellets were resuspended in complete culture medium (neurobasal containing 2% B27) and seeded on poly-L-lysine (Sigma-Aldrich, USA) precoated 96-well plates. Cells were cultured in a humidified atmosphere (5% CO2/95% room air) at 37 C for 2 h, and then the medium was replaced with neurobasal medium containing 1% penicillin/streptomycin, 2% B27, and 2 mM Gluta-MAX-I supplements. The primary neurons were ready for use 7 days after seeding. Animals. SD rats and ICR mice were obtained from Shanghai SLAC Laboratory Animal (Shanghai, China). The animals were housed in a specific pathogen-free animal facility with free access to food and water. The protocol for animal experiments was approved by the Animal Experimentation Ethics Committee of Shanghai Jiao Tong University School of Medicine. Preparation and Characterization of GM1-rHDL and rNAP-GM1-rHDL. rHDL- and DiI-loaded rHDL was prepared as previously described.16 GM1-DMPC liposomes were prepared as follows: 3.6 mg of DMPC and 0.4 mg of GM1 were first dissolved in a solvent mixture of methanol/chloroform (1:2, v/v). The solution was then vacuum-dried with the lipid film rehydrated in 4 mL of 0.01 M PBS buffer (pH 7.4). The obtained turbid emulsion was subsequently bath sonicated at 40 C for 1 h and then probe sonicated (Scientz Biotechnology, China) at 200 W output for 15 min. For preparing GM1-rHDL, apoE3 (0.8 mg) was then added into 4 mL of 1 mg/mL GM1-DMPC-liposome emulsion and incubated at 37 C for 36 h. To prepare RNAP-GM1-rHDL, RNAP was dissolved in deionized water, added into GM1-DMPC liposomes at a weight ratio of 1:125 (RNAP:DMPC), and incubated at 4 C overnight. Then apoE3 (0.8 mg) was added into the above emulsion and incubated at 37 C for 24 h. The free RNAP

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GM1 incorporated into the lipid membrane, GM1-rHDL demonstrated subnanomolar magnitude and much higher Aβ1
42 binding affinity than rHDL, more efficiently accelerated microglia-mediated cellular Aβ1
42 degradation, and also facilitated the BBB efflux of Aβ1
42. Following the noninvasive intranasal administration, GM1-rHDL exhibited higher brain biodistribution efficiency than rHDL. To justify the potential of GM1-rHDL as a multifunctional nanostructure, the neuroprotective peptide NAP was chosen as a model drug to be loaded into GM1-rHDL following its fusion with R-helix peptide. The obtained RNAP-GM1-rHDL

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volume of Soluene (PerkinElmer, USA) at 60 C for 1 h, then added with 3.6-fold volumes of Hionic-Fluor (PerkinElmer, USA) and analyzed via a liquid scintillation counter. The BEI (%) was defined as56 BEI (%) ¼

drug effluxed at the BBB  100% drug injected into the brain

and calculated as 2

3 amount of Aβ remaining in the brain 6 amount of inulin remaining in the brain7 7 BEI (%) ¼ 6 41
5 amount of Aβ injected amount of inulin injected

ARTICLE

GM1-rHDL (0, 0.01, 0.05, 0.5, or 2 μg/mL) at 37 C for 4 h. After that, the cells were washed with PBS, fixed with 3.7% formaldehyde solution, and stained with Hoechst 33258. The cells were finally washed three times with PBS, and the cellular uptake of FAM-Aβ1
42 was quantitatively analyzed using an HCS instruments (Thermo Scientific Cellomics, Thermo, USA) as described previously.16 Intracellular Aβ Degradation Assay. Primary microglia were plated at a density of 1.2 105 cells/well in a 24-well plate. Twentyfour hours later, the cells were incubated with Aβ1
42 (2 μg/mL) in serum-free medium in the presence of rHDL or GM1-rHDL (0, 1, 10, or 100 μg/mL) at 37 C for 4 h. After washing with PBS, the cells were lysed in 1% SDS containing a protease inhibitor cocktail (Roche, Switzerland). The total protein content of cell lysates was analyzed by a bicinchoninic acid (BCA) protein assay (Thermo, USA). The remaining intracellular Aβ1
42 levels were quantified with an ELISA kit (Invitrogen, USA) and normalized to total protein of the lysates. Colocalization Assay. For evaluation of the colocalization between DiI-rHDL/GM1-rHDL and FAM-Aβ1
42, primary microglia were plated at a density of 1 104 cells/well in a 96-well glassbottom plate in DMEM containing 10% FBS and cultured for 24 h to allow cell attachment. After that, the cells were treated with 2 μg/mL FAM-Aβ1
42 in the presence of DiI-rHDL/DiI-GM1rHDL (10 μg/mL) at 37 C for 4 h. Cellular distribution of FAMAβ1
42 and DiI-rHDL/DiI-GM1-rHDL was analyzed with Thermo Scientific Cellomics Colocalization software. For evaluation of the colocalization between DiO-GM1-rHDL and lysosome, primary microglia were plated at a density of 2  104 in a 24-well glass bottom culture plate as described above for 24 h and then treated with DIO-GM1-rHDL (10 μg/mL) in serum-free DMEM at 37 C for 4 h with LysoTraker Red DND-99 (Invitrogen, USA), an indicator of lysosome, added and incubated for 0.5 h. After that, the cells were fixed and stained with Hoechst 33258, and the cellular distribution of DIO-GM1-rHDL was analyzed with Thermo Scientific Cellomics Colocalization software. Brain Distribution of GM1-rHDL/rHDL Following Intranasal Administration. GM1-rHDL and rHDL were labeled with 125I according to the Bolton
Hunter procedure as described previously.48 The 125 I-rHDL and 125I-GM1-rHDL were stored at 4 C before the experiment and used within 24 h, and the stability of 125I-labeling on both rHDL and GM1-rHDL was confirmed to be more than 90% via a thin-layer chromatography analysis. For biodistribution analysis, male ICR mice (25 ( 2 g) were nasally dosed with a bolus of 125I-rHDL or 125I-GM1-rHDL (25 μCi/mice in 10 μL, at the lipid dose of 5 mg/kg). For the administration, conscious mice were fixed in a prostrate position, and the preparations were given at the openings of the nostrils via a polyethylene 10 tube attached to a microsyringe. The procedure lasted about 4 min, allowing the animal to inhale all of the preparations. At designated time points (0.167, 0.5, 1, 2, 4, 8, and 12 h after the nasal administration), blood was collected, and the mouse was sacrificed immediately, with brain sections and peripheral tissue samples collected, weighed, and assayed for radioactivity. Brain Aβ1
42 Clearance in Mice. Aβ1
42 oligomer was labeled with 125I via the Iodogen iodination method as previously discribed.80 The 125I-Aβ1
42 and [14C]inulin (PerkinElmer, USA) were mixed and diluted with artificial CSF to reach a final radioactivity of 0.05 and 0.08 μCi/μL, respectively. For the analysis, male ICR mice (25 ( 2 g) were randomly divided into three groups and nasally administrated with 10 μL of PBS, rHDL, and GM1-rHDL (at a lipid dose of 5 mg/kg), respectively. Thirty minutes later, the mice were anesthetized by intraperitoneal injection of chloral hydrate, fixed in a stereotaxic frame, and intrahippocampally injected via a microsyringe with 0.6 μL of the mixture of 125I-Aβ1
42 (0.28 ng per mice) and [14C]inulin over 2 min, at the following coordinates: 2.3 mm posterior to the bregma, (1.8 mm lateral to the midline, and 2.0 mm ventral to the skull surface. At the time points of 10, 30, and 90 min after the intrahippocampal injection, the mice were sacrificed, and the cerebrum collected, divided into two parts, and weighed. One part of the cerebrum was analyzed via a γ-counter, then homogenized and precipitated in 3-fold weight of cold TCA to determine the intact Aβ1
42; the other part of the cerebrum was homogenized in deionized water and solubilized in 10-fold

 100% The 100  (Nb/Ni) was defined as55 100  (Nb =Ni ) amount of TCA precipitated Aβ remaining in the brain ¼ amount of Aβ injected 100% Neurite Outgrowth Assay. Primary cultured neurons were seeded in a 96-well plate at a density of 1.5  104 cells/well. Seven days after seeding, the neurons were treated with 10 μM Aβ1
42 oligomers or 200 μM glutamic acid (Sigma-Aldrich, USA) in the presence of RNAP, GM1-rHDL, or RNAP-GM1-rHDL at 37 C for 48 h, fixed in 3.7% formaldehyde, and then sequentially stained with MAP2 primary antibody (Merk Millipore, Germany) and Alexa Flour488-conjugated secondary antibody (Invitrogen, USA) for the visualization of neuronal cell bodies and neurites and with Hoechst 33342 for nuclei detection. The stained cells were finally scanned under the HCS instrument and analyzed via the Thermo Scientific Cellomics Neuronal Profiling Bioapplication software. Aβ1
42 and IBO Co-injection AD Mice Model. Aβ1
42 was first dissolved in artificial CSF (2 mg/mL) and allowed to aggregate following the incubation at 37 C for 4 days. Before the coinjection, Aβ1
42 and IBO (Sigma-Aldrich, USA) solutions (dissolved in artificial CSF, 1 mg/mL) were first mixed to a final concentration of 1 μg/μL Aβ1
42 and 0.5 μg/μL IBO. Male ICR mice were anaesthetized with chloral hydrate and then fixed in a stereotaxic frame. The animals were bilaterally injected into the dorsal hippocampus (2.3 mm posterior to the bregma, (1.8 mm lateral to the midline, and 2.0 mm ventral to the skull surface) via a microsyringe for over 2 min with 2 μL of the mixture of Aβ1
42 and IBO to establish the AD model mice, with animals injected with the same volume of artificial CSF as the sham control. All the mice were allowed to recover for 2 days before drug treatment. Drug Treatment of AD Model Mice. AD model mice (n = 7 or 8 per group) were daily nasally treated with 10 μL of rHDL, GM1-rHDL, or RNAP-GM1-rHDL at a lipid dose of 5 mg/kg or RNAP solution at a dose of 24 μg/kg for 2 weeks, respectively. As negative and normal controls, AD model mice and sham control mice were nasally treated with 10 μL of PBS daily for 2 weeks. MWM Test. The MWM setting consisted of a circular pool (diameter, 150 cm; height, 50 cm) equipped with a 9 cm platform 1 cm below the surface of the opacified water (30 cm deep) in the middle of a quadrant. On the first 5 days, the animals were trained four times daily from four different positions around the border of the maze with different sequences on each day. The cutoff time for the latency to reach the platform was 60 s. If an animal failed to reach the platform within 60 s, it was guided to the platform and kept there for 30 s. The swimming path and escape latencies were recorded using a tracking system (Shanghai Jiliang Software Technology, China). The probe trails were performed on the sixth day with the platform removed, and the mice were placed in water from the two starting points away from the platform by order. Spatial acuity was expressed as the percentage of time the animal spent in the quadrant where the escape platform used to be located. Immunohistochemical Analysis. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded

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Conflict of Interest: The authors declare no competing financial interest. Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b03124. Biodistribution of 125I-rHDL and 125I-GM1-rHDL following intranasal administration; colocalization analysis between GM1-rHDL and lysosomes; neuroprotective effects of RNAP on Aβ1
42 oligomer-induced neuronal toxicity; tissue samples of AD model mice treated with RNAP solution, rHDL, GM1-rHDL, and RNAP-GM1-rHDL (PDF) Acknowledgment. This study was supported by National Natural Science Foundation of China (Nos. 81373351, 81573382), grants from Shanghai Science and Technology Committee (12NM0502000 and 14ZR1423700), Shanghai Talent Development Fund, National Science and Technology Major Project 2012ZX09303001-001, and SJTU Funding YG2014MS75.

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