SCIENCE ADVANCES | RESEARCH ARTICLE

HEALTH AND MEDICINE Copyright © 2020

The Authors, some
Blood-brain barrier–penetrating siRNA nanomedicine rights reserved;
exclusive licensee
for Alzheimer’s disease therapy American Association
for the Advancement
of Science. No claim to
Yutong Zhou1*, Feiyan Zhu2*, Yang Liu2,3*, Meng Zheng2†, Yibin Wang2, Dongya Zhang2, original U.S. Government
Yasutaka Anraku4, Yan Zou2,5, Jia Li3, Haigang Wu2, Xiaobin Pang3, Wei Tao6, Olga Shimoni7, Works. Distributed
Ashley I. Bush8, Xue Xue1†, Bingyang Shi2,5† under a Creative
Commons Attribution
Toxic aggregated amyloid- accumulation is a key pathogenic event in Alzheimer’s disease (AD), which derives NonCommercial
from amyloid precursor protein (APP) through sequential cleavage by BACE1 (-site APP cleavage enzyme 1) and License 4.0 (CC BY-NC).
-secretase. Small interfering RNAs (siRNAs) show great promise for AD therapy by specific silencing of BACE1.
However, lack of effective siRNA brain delivery approaches limits this strategy. Here, we developed a glycosylated
“triple-interaction” stabilized polymeric siRNA nanomedicine (Gal-NP@siRNA) to target BACE1 in APP/PS1
transgenic AD mouse model. Gal-NP@siRNA exhibits superior blood stability and can efficiently penetrate the
blood-brain barrier (BBB) via glycemia-controlled glucose transporter-1 (Glut1)–mediated transport, thereby
ensuring that siRNAs decrease BACE1 expression and modify relative pathways. Noticeably, Gal-NP@siBACE1 ad-
ministration restored the deterioration of cognitive capacity in AD mice without notable side effects. This “Trojan
horse” strategy supports the utility of RNA interference therapy in neurodegenerative diseases.

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INTRODUCTION clinical trial (9). However, several BACE1 small-molecule inhibitors
Alzheimer’s disease (AD) is the most common age-related neuro- have been shelved by pharmaceutical companies due to off-target
degenerative disorder, characterized by progressive deterioration of toxicity and other safety reasons (10, 11). Despite these recent failures,
cognitive capacity (1). In 2019, AD affected more than 50 million BACE1 is still considered as one of the most promising therapeutic
people globally, which is expected to reach 152 million by 2050 (2). targets for AD (8, 11).
In addition, the current annual cost of AD worldwide is $1 trillion, Compared to small molecule–based approaches, small interfer-
which is estimated to double by 2030 (2). Currently, clinical therapy ing RNAs (siRNAs) offer promising therapeutics for brain disease
using acetylcholinesterase inhibitors or N-methyl-d-aspartate receptor treatment by directly blocking causative gene expression with high
antagonists are palliative treatment options, which only moderately targeting specificity, low effective doses, and a relatively simple
improve cognition and behavior in Alzheimer’s patients but do not drug development process (12). siRNA in a lentiviral vector silenc-
slow disease progression (3, 4). Hence, it is imperative to develop ing BACE1 has also been shown to ameliorate AD neuropathology
therapeutics targeting pathological mechanisms in AD. (13). However, effective and safe systemic delivery of siRNA into the
The precise pathological mechanisms leading to AD are not fully brain remains challenging, reflecting the presence of biological
understood. However, plaques composed of aggregated amyloid- barriers such as the blood-brain barrier (BBB), short circulation
peptide (A), neurofibrillary tangles containing hyperphosphorylated lifetime, enzymatic degradation, insufficient tissue penetration, cell
tau protein, and neuroinflammation are pathological hallmarks (5). endocytosis, and impaired cytosolic transport. Recent studies have
Among these, the aberrant accumulation of A resulting from the shown that nanodelivery approaches hold great potential for over-
sequential cleavage of the amyloid precursor protein (APP) by coming these challenges (12). It has been reported that the delivery
BACE1 (-site APP cleavage enzyme 1) and -secretase activity is of BACE1 siRNA (siBACE1) to the mouse brain by systemic injec-
believed to be a key pathogenic event in AD (6). As a result, strate- tion can partially reduce AD neuropathology (14, 15). However, the
gies that reduce BACE1 activity, and thereby A levels, have been therapeutic efficacy was less than ideal probably due to low siRNA
considered as a potential therapeutics for AD (7, 8). A skin patch brain accumulation and poor stability. In this work, we report an
consisting of BACE1 inhibitor therapeutics has entered phase 3 effective, nonviral, and BBB-penetrable siBACE1 nanodelivery
approach that we evaluate in a well-established AD mice model that
1
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai
offers the potential for clinical translation.
University, Tianjin 300350, China. 2Henan-Macquarie Uni Joint Centre for Biomedical We developed a glycosylated nanodelivery system, which uses
Innovation, School of Life Sciences, Henan University, Kaifeng, Henan 475004, China. glycemia-controlled Glut1 (glucose transporter-1) recycling to facilitate
3
School of Pharmacy, Henan University, Kaifeng, Henan 475004, China. 4Depart- nanomedicine BBB penetration for more effective AD therapy (Fig. 1).
ment of Bioengineering, Graduate School of Engineering, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. 5Department of Biomedical To improve biophysiological protection of the encapsulated siRNA, we
Sciences, Faculty of Medicine & Health Sciences, Macquarie University, Sydney, used a “triple-interaction” stabilization method reported previously (16).
NSW 2109, Australia. 6Center for Nanomedicine and Department of Anesthesiology, Specifically, we make use of a guanidinium-phosphate (Gu+/PO34−) salt
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA.
7
Institute for Biomedical Materials & Devices (IBMD), School of Mathematical and
bridge to provide a stabilizing electrostatic and hydrogen bond inter-
Physical Sciences, University of Technology Sydney, 15 Broadway, Ultimo, NSW action, in addition to the hydrophobic interaction, which is derived
2007, Australia. 8Florey Institute of Neuroscience and Mental Health, University of by the complexation between siRNA and the galactose-­modified
Melbourne, Melbourne, VIC 3010, Australia. poly(ethylene glycol)-block-poly[(N-(3-methacrylamidopropyl)
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected] (B.S.); [email protected] (X.X.); guanidinium [Gal-PEG-b-P(Gu)]/poly(ethylene glycol)-­block-poly
[email protected] (M.Z.) [(N-(3-methacrylamidopropyl) guanidinium-co-2,2,3,3-tetrafluoropropyl

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SCIENCE ADVANCES | RESEARCH ARTICLE

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Fig. 1. Illustration of the formation of the glycosylated “triple-interaction” stabilized siRNA nanomedicine (Gal-NP@siRNA) and the mechanism and approach
to treat AD pathology in APP/PS1 transgenic mice. (A) Schematic illustration of the fabrication of Gal-NP@siRNA. (B and C) Mechanism by which Gal-NP@siRNA pene-
trates the BBB and accumulates in the brain. Glut1 is overexpressed on the luminal membrane of the BBB after 24-hour fasting. After treatment with Gal-NP@siRNA, glu-
cose replenishment in fasting mice results in Glut1 recycling from the luminal to the abluminal membrane of the BBB, which leads to the transport of Gal-NP@siRNA across
the BBB. (D) Gal-NP@siRNA–mediated knockdown of BACE1 mRNA expression, which leads to reduced levels of amyloid plaques.

methacrylate] [PEG-b-P(GuF)] polymer mixture. Our triple-interaction and fig. S1). Because of the hydrophobic interaction of the fluorine
stabilized siRNA nanomedicine demonstrates superior stability per- in P(GuF) for nucleic acid stabilization enhancement (16, 24), gel
formance in blood circulation relative to conventional cationic retardation assays demonstrated that the Gal-PEG-b-P(Gu)/PEG-
polymer-based nanomedicines that feature only a single electrostatic b-P(GuF) polymer mixture can more effectively encapsulate siRNA
interaction (16). Furthermore, exploiting Glut1 recycling by initially compared to the fluorine-free Gal-PEG-b-P(Gu) polymers (complete
inducing hypoglycemia, which elevates Glut1 expression on the siRNA loading weight ratio: 2.5:1 versus 10:1) (Fig. 2A and fig. S2A).
luminal plasma membrane of the BBB, facilitates markedly enhanced Moreover, fluorinated nanomedicines showed better performance in
delivery of glucose-modified nanocarriers across the BBB when stability assays compared to fluorine-free counterparts in the negatively
Glut1 is recycled to the abluminal membrane of the BBB upon glucose charged biomacromolecule heparin competition assay (fig. S2B),
replenishment (17–19). To facilitate the Glut1 recycling approach, signifying the importance of fluorination to improve the stability of
we appreciated that Glut1 stereochemistry allows binding of both siRNA nanomedicines. These siRNA nanoparticles (NPs) were then
d-glucose and d-galactose (20–23). We thus hypothesized that our characterized by dynamic light scattering (DLS) and transmission
galactose-modified siRNA nanomedicines should bind to Glut1 to electron microscopy (TEM) (Fig. 2, B and C). These results showed
efficiently penetrate the BBB by glycemia-controlled Glut1-mediated that Gal-NP@siRNA nanomedicine exhibited a spherical morphology
transport. Consequently, we demonstrate that siBACE1 glycosylated with an average size of 118 nm and a low polydispersity index of
siRNA nanomedicine is efficiently delivered to the brains of APP/ 0.13 at a polymer/siRNA mass ratio of 2.5:1. Moreover, the nano-
PS1 transgenic mice and ameliorates AD-like pathology, leading to medicine exhibited excellent stability in both phosphate-buffered
improvement in cognitive impairment. saline (PBS) and 10% fetal bovine serum (FBS) (fig. S2C).
A key point for siRNA nanodelivery for AD therapy is an effec-
tive neural cell endocytosis and cytosolic transport. Flow cytometry
RESULTS analysis and confocal imaging showed that both glycosylated and
Biophysical characterization and in vitro studies nonglycosylated siRNA nanomedicines are efficiently taken up by
of Gal-NP@siRNA Neuro-2a cells (Fig. 2, D and E). The Gal-NP@siRNA nanomedicine
In this study, the glycosylated triple-interaction stabilized siRNA also displayed effective endosome escape ability (fig. S3). In addition,
nanomedicine (Gal-NP@siRNA) was prepared by complexation competitive cellular binding assay of Gal-NP@Cy5-siRNA in general
between siRNA and Gal-PEG-b-P(Gu)/PEG-b-P(GuF), which were Glut1 inhibitor phloretin treatments showed a dose-dependent uptake
synthesized by reversible addition-fragmentation chain transfer co- in Glut1 highly expressed cells (fig. S4), which is consistent with
polymerization of hydrophobic monomer 2,2,3,3-tetrafluoropropyl previous report (18), indicating Glut1 as the dominant endocytosis
methacrylate and siRNA complexation segment N-(3-methacryl- pathway. Next, to quantify the efficiency of siRNA silencing of the
amidopropyl) guanidinium (Gu) (for detailed synthesis, see scheme S1 target gene, BACE1 mRNA and protein expression were examined

Zhou et al., Sci. Adv. 2020; 6 : eabc7031 9 October 2020 2 of 14

SCIENCE ADVANCES | RESEARCH ARTICLE

A B 16 C
Polymer:siRNA

Intensity (%)
12
0 1 2.5 5 10 15 20

0
101 102 103
Size (nm)

D E
DAPI FAM TRITC Merge

160 PBS Free siRNA

NP@siRNA Gal-NP@siRNA
Free

140

120

100

Count

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NP@siRNA

80

60

40

20

0
@siRNA

102 103 104 105

Gal-NP

Cy5-siRNA fluorescence intensity

F 1.2 *** *** G

BACE1 relative mRNA

1.0
expression level

0.8

0.6 (kDa)
-70
0.4
BACE1

0.2
GAPDH -35
0.0

Fig. 2. Biophysical characterization and in vitro studies of Gal-NP@siRNA. (A) Gel retardation assay of Gal-NP@siRNA at polymer/siRNA weight ratios of 1, 2.5, 5, 10,
15, and 20. (B) Size distribution and (C) transmission electron micrographs of Gal-NP@siRNA. (D) Confocal laser scanning microscopy images for NP cellular uptake. Images
were collected for Neuro-2a cells after 4-hour NP incubation. Cell nuclei were stained with DAPI (blue), siRNA was labeled by FAM dye (green), and cell cytoskeleton was
stained with TRITC-phalloidin (red) to indicate cytoplasm area. Scale bars, 10 m. (E) Flow cytometry analysis of Neuro-2a cells following 4-hour incubation with free
Cy5-siRNA, NP@Cy5-siRNA, and Gal-NP@Cy5-siRNA. (F and G) In vitro gene silencing effects of Gal-NP@siBACE1 and controls at day 3 post transfection. BACE1 mRNA (F)
and protein (G) expression levels was quantified by qRT-PCR and western blot assay, respectively. Data are presented as mean ± SEM (n = 3, ***P < 0.001).

in Neuro-2a cell samples treated with Gal-NP@siBACE1. The results no obvious toxicity in multiple neuron-related cells (fig. S5). In
showed that the BACE1 gene was sufficiently silenced in Neuro-2a contrast, NPs loaded with scrambled siRNA (siScr) failed to reduce
cells (Fig. 2, F and G), achieving approximately 46 and 45% BACE1 BACE1 mRNA and protein levels, corroborating the sequence-­
mRNA and protein down-regulation, respectively, while exhibiting specific gene silencing activity of siBACE1. The superior silencing

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SCIENCE ADVANCES | RESEARCH ARTICLE

ability of Gal-NP@siBACE1 in Neuro-2a cells likely reflects their Experimental nesting data showed that Gal-NP@siBACE1–
stable encapsulation and siRNA protection, efficient cellular inter- treated APP/PS1 mice achieved a similar score to WT mice,
nalization, and endosome escape, characteristics that show promis- which was much better than all other APP/PS1 control groups
ing potential for in vivo study. (Fig. 4, B and C). Furthermore, the NOR test results showed that
PBS-treated APP/PS1 control mice showed suppressed interest in
Biodistribution and in vivo BACE1 targeting efficacy exploring novel objects compared with WT mice as determined by
of Gal-NP@siRNA discrimination index (DI) and preference index (PI) for novel
Next, to evaluate in vivo pharmacokinetics, the plasma levels of object (Fig. 4, D to F). After being treated with Gal-NP@siBACE1,
Cy5-labeled siRNA were measured after intravenous injection of APP/PS1 mice showed a significant increase in NOR compared
free siRNA, fluorinated siRNA nanomedicine, and fluorine-free siRNA to PBS-treated APP/PS1 control mice. Excitingly, the DI and PI
nanomedicines. These data demonstrated that the fluorinated siRNA for novel object reached the performance of normal WT mice
nanomedicine (Gal-NP@siRNA) had the longest blood circulation (Fig. 4, E and F). In contrast, control APP/PS1 mice treated with
time with an elimination half-lifetime (t1/2) of 39.2 min (Fig. 3A), non–galactose-modified NP@siBACE1 or Gal-NP@siScr performed
which was significantly longer than that of both the fluorine-free as poorly as PBS-treated control APP/PS1 mice, signifying the
counterpart and free siRNA (t1/2 of 22.0 and 8.0 min, respectively; importance of the targeting ability of the galactose ligand and the
fig. S6A and Fig. 3A). These circulation results were consistent with therapeutic effect of siBACE1 brain delivery. In the MWM test, all
the data above demonstrating biophysical stability and good hepa- groups achieved comparable escape latencies (fig. S7) during the
rin competition characteristics, further confirming the excellent five training days.
stability of fluorinated siRNA NPs. Subsequently, we studied the On the probe test day, when the escape platform was removed,
in vivo brain targeting of our glycosylated siRNA nanomedicine by long-term spatial memory has been investigated (Fig. 4, G to J). How-

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glycemia-controlled Glut1-mediated transport. The biodistribution ever, on probe test day, mice administered with PBS, NP@siBACE1,
of Gal-NP@Cy5-siRNA was quantified by fluorometry. These ex- and Gal-NP@siScr showed an aimless searching strategy with no or
periments demonstrated that the brain accumulation of Gal-NP@ only slightly improved spatial learning and memory (see representa-
Cy5-siRNA nanomedicine was up to 5.8-fold higher than that of tive tracking plots in Fig.  4G), with reduced time in the target
non–galactose-modified NP@Cy5-siRNA nanocarriers (Fig. 3B and quadrant but similar swimming speed compared to WT controls
fig. S6B). In addition, we also observed that the siRNA brain accu- (Fig. 4, H and I). In contrast, APP/PS1 mice treated with Gal-NP@
mulation reached a peak at 1 hour after injection and maintained a siBACE1 exhibited a greater proportion of time in the target
considerable fluorescence accumulation up to 24 hours, as monitored quadrant and number of platform crossings compared to PBS-­
by Cy5 in vivo imaging (Fig. 3C), and the BACE1 mRNA and protein injected controls (Fig. 4, I and J). These data confirm that the
expression in cortex was inhibited after nanomedicine treatment Gal-NP@siBACE1 nanomedicine mediates highly effective siRNA
(Fig. 3, D and E, and fig. S6C). brain delivery to significantly improve cognitive performance in
APP/PS1 mice.
Behavioral evaluation of Gal-NP@siBACE1 nanomedicine
therapy in APP/PS1 mice Effects of the Gal-NP@siBACE1 treatment on APP processing
To evaluate the therapeutic effect of Gal-NP@siBACE1 in a relevant and amyloid deposition in APP/PS1 mice
AD pathology model, the APP/PS1 double transgenic mouse model After behavioral tests were completed, mice were sacrificed, and
was assessed in behavioral tests of learning and memory impair- brain tissue was collected for analysis of BACE1 suppression and its
ment relevant to AD. The APP/PS1 double transgenic mouse is a impact on A and tau pathological accumulation (Fig. 5A). Our
commonly used multitransgenic animal model that expresses two results showed that both hippocampal and cortical BACE1 protein
familial AD mutant genes for APP together with mutant presenilin levels in Gal-NP@siBACE1–treated APP/PS1 mice were significantly
1 (PS1). Compared to single transgenic mice and other nongenetic decreased compared to other APP/PS1 control groups (Fig. 5B and
AD mouse models, APP/PS1 mice express accelerated amyloid fig. S8, A and B), in agreement with the improvement in behavioral
deposition and synaptic loss with reliable memory deficits (25–27). tests. Hence, effective BACE1 protein silencing shown by Gal-NP@
BACE1 inhibition has been reported to prevent neuron loss and siBACE1 demonstrates a reliable siRNA delivery approach for
memory deficits in APP/PS1 mice (28), demonstrating suitability for targeting the brain. The manifestation of pathological hallmark
evaluating the therapeutic efficacy of novel nanocarriers in this model. of AD, amyloid plaques derived from BACE1-cleaved APP, was
To determine whether Gal-NP@siBACE1 nanomedicine could significantly decreased with reduced foci size in both the hippo-
ameliorate neuropathology in APP/PS1 transgenic mice, APP/PS1 campus and cortex of Gal-NP@siBACE1–treated APP/PS1 mice
mice were given Gal-NP@siBACE1 or control Gal-NP@siScr (siRNA, (Fig. 5, C and D). In sharp contrast, control PBS–, NP@siBACE1-,
1 mg/kg) via caudal vein injection every 3 days (Fig. 4A). The same or Gal-NP@siScr–treated mice exhibited pronounced A plaque
dose of non–galactose-modified NP@siBACE1 was assessed as a deposition (Fig. 5, C and D). Another major pathological feature
negative control. PBS-injected APP/PS1 and control wild-type (WT) of late-stage AD is the development of intracellular neurofibrillary
mice groups were included to ascertain AD-relevant deficits in tangles composed of hyperphosphorylated tau protein (p-tau),
APP/PS1 mice at baseline. Behavioral tests including the novel which synergistically impairs cognitive performance in AD patients
object recognition (NOR) test and the Morris water maze (MWM) (33). Our data showed that both hippocampal and cortical p-tau
were performed to examine spatial learning and memory, while the levels in AD mice treated with Gal-NP@siBACE1 were lower than
nest-building test was used to assess general health and hippocampal those in control AD mice treated with PBS (Fig. 5E and fig. S8C),
function, because nest building is often impaired in rodent models probably attributable to the molecular interplay between A and
of AD (29–32). p-tau (34, 35).

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SCIENCE ADVANCES | RESEARCH ARTICLE

A ( 103)
120 Free siRNA, t1/2. = 8.0 min C 6.0
100 Gal-NP@siRNA, t1/2. = 39.2 min

siRNA con. (%) 80

60
5.0
40
20 0h 0.5 h 1h
0
4.0
0 100 200 300 400
Time (min)

B
25 NP@siRNA 3.0
Gal-NP@siRNA
20 NP@siRNA Gal-NP@siRNA ( 107)
16.0 2h 4h 6h
15
% ID/g

10 14.0 2.0

5 *
12.0
0

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Radiant efficiency Min = 1.11e8
Counts
Max = 1.72e8
Min = 1067
8h 12 h 24 h Max =6161

D 1.4 1.4
BACE1 relative mRNA

1.2 1.2
expression level

BACE1/GAPDH
1.0 1.0
* E **
0.8 * 0.8
0.6 (kDa) 0.6
0.4 BACE1 -70 0.4
0.2 0.2
GAPDH -35
0.0 0.0 s S

G siB cr
A
s S

-N P@ E1

A r
E1
G siB cr
N P@ A

-N P@ E1

E1
A r

si iSc
si iSc

ee PB
ee P B

N iRN
N iRN

iS
iS

C
C

P @ @s

P@ s
A
P@ s

P@ s
A

B
B

N
al N

Fr
Fr

G al-
G al-

al

Fig. 3. Biodistribution and in vivo BACE1 targeting efficacy of Gal-NP@siRNA. (A) In vivo pharmacokinetics as shown by Cy5-siRNA concentration/time curves in
plasma after a single-dose injection. (B) (Left) Quantification of Cy5-siRNA accumulation in different organs. Cy5-siRNA levels were determined by fluorescence spectros-
copy 1 hour after tail vein injection of siRNA nanomedicine after a single-dose injection. Data are presented as mean ± SEM (n = 3, *P < 0.05). (Right) Representative image
for Cy5 signal in the brain of NP@siRNA and Gal-NP@siRNA groups 1 hour after injection. (C) Time course in vivo imaging of Gal-NP@Cy5-siRNA evaluated by fluorescence
imaging after a single-dose injection. (D and E) BACE1 mRNA and protein expression level in cortex was quantified by (D) qRT-PCR and (E) Western blot assay from WT
mice samples, and samples were collected at day 3 after two nanomedicine treatments. Data are presented as mean ± SEM (n = 3, *P < 0.05).

BACE1 deficiency can impair remyelination, which negatively Cytotoxicity and in vivo biocompatibility assessment
affects cognitive function (36). Therefore, it is imperative to knock of the Gal-NP@siRNA nanomedicine
down BACE1 to an appropriate therapeutic level. To check this, we To further assess the biocompatibility and systemic response to the
followed the expression of the multilamellar myelin sheath for- nanomedicine, we assessed routine blood parameters and chemistry
mation marker myelin basic protein (MBP) in the central nervous by measuring plasma alanine aminotransferase (ALT), aspartate
system (CNS). In PBS-treated control APP/PS1 mice, the expres- aminotransferase (AST), alkaline phosphatase (ALP), plasma urea
sion of MBP in the brain was significantly decreased (Fig. 5F and (BUN), uric acid (UA), creatinine (CR), as well as blood platelet
fig. S8D), indicating the negative consequences of A deposition on (PLT), red blood cells (RBCs), and white blood cells (WBCs)
myelin, which was consistent with reported literature (37). In con- (Fig. 6, A and B, and fig. S9A). To evaluate the inflammatory concern
trast, MBP protein expression was restored in AD mice treated of the nanomedicine treatments, core proinflammatory cytokines
with Gal-NP@siBACE1 to levels seen in WT mice (Fig. 5F), thereby such as Il-1, Il-6, and Tnf- have been tested in liver and kidney
indicating that the dose of siBACE1 was appropriate and sufficient (Fig. 6, C and D). These examinations demonstrated no significant
to neutralize A toxicity. difference between PBS and Gal-NP@siRNA treatment groups

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SCIENCE ADVANCES | RESEARCH ARTICLE

A
siRNA, 1 mg/kg every 3 days Therapeutic evaluation

8-month-old 10 times treatments (i.v.) 21 3 4 5 6 7 8 9 10 11 12 (day)

APP/PS1 Nest NOR MWM
construction
Sacrifice

B 4 **
WT-PBS AD-PBS AD-NP@siBACE1 C

Scores in nesting
3 ** **
2

AD-Gal-NP@siScr AD-Gal-NP@siBACE1 Point paper towels Bites

0
0 No
1 Throughout the cage
2 Concentrated in the cage
3 Concentrated on one side
4 Gathered together

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D E * F
Discrimination index

Training Testing 0.6

* * 0.8

Preference index
0.4
0.6 ** **
0.2
24 h 0.0 0.4
–0.2
0.2
–0.4
–0.6 0.0

G WT-PBS AD-PBS AD-NP@siBACE1 AD-Gal-NP@siScr AD-Gal-NP@siBACE1

: Target
: Pool quadrant

: Escape : Motion
platform path

H I
60
** J 8 ** *
Time in the targeted

Number of platform

40 **
quadrant (%)

6
speed (cm/s)

crossings

40
Swimming

30
4
20
20
10 2

0
0 0

Fig. 4. Behavioral evaluation of Gal-NP@siBACE1 nanomedicine therapy in APP/PS1 mice. (A) Schematic of the experimental timeline. APP/PS1 and WT mice were
treated with siRNA nanomedicine or PBS via tail vein injection every 3 days (10 cycles). Mice were then subjected to nesting, NOR, and MWM tests for memory evaluation,
and samples for molecular pathological assessments were collected. (B) Representative images and scoring criteria from the nest-building experiment in APP/PS1 and
control WT mice. Photos were taken 24 hours after the introduction of nesting material to the home cage. Photo credits: Yutong Zhou, Nankai University. (C) Nest-building
scores for each group. (D) Setup for NOR test. (E and F) Results for NOR test. (E) DI and (F) PI of each group after nanomedicine treatment. (G to J) Data for probe test in
the MWM. (G) Representative swimming track, (H) swimming speed, (I) ratio of time spent in target quadrant, and (J) number of crossing the platform location of each
group on the probe test day. All behavioral test bar or plot charts are presented as mean ± SEM (n = 6 to 8, *P < 0.05, **P < 0.01).

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SCIENCE ADVANCES | RESEARCH ARTICLE

WT AD
A B

Cortex Hippocampus
(kDa)
-70
BACE1
ACTB -40

BACE1 -70

ACTB -40

** ** * *
** *

C
Hippocampus

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D **
** ** **

Plaque burden (%)

** **
Cortex

A NP AD BS
-G a iB BS

B cr
P@ @ 1

E1
-N P CE
si iS
C
P
A D- @ -P

s
al l-N A

A
T-
W

D G s
-
D
A
WT AD
WT AD
E F
Hippocampus

Cortex Hippocampus

(kDa)
p-Tau (kDa)
-55 MBP -15
ACTB -40 ACTB
-40
-55 -15
Cortex

p-Tau MBP
ACTB -40 ACTB -40

Cortex
**
**
* * 4 **
** **
** **
* * ** **
3 ** **
p-Tau/ACTB

MBP/ACTB
MBP/ACTB

0
A AD P@ AD BS

1
al l - N B A S

B cr
E1

A AD P@ AD BS
al l-N BA S

1
A AD P@ AD BS
al l-N BA S

A AD P@ AD BS
AC r

al l-N A S
E1
A r
1

A r
E1

E1
B c

B c
B c
P@ @ E

P@ @ E

P@ @ E
P@ @ E
-G a si -PB

-G a si -PB

-G a si -PB
-G a si -PB
si siS

si siS

si siS
si siS

-N P C
-N P C

C
C

-N P C

C
-N P C

P
P
P

P
A

T-
T-
T-

T-

B
W
W
W

-N

-N
D -G

D -G
-N

-N
D -G

D -G

D
D

A
A

Fig. 5. Therapeutic evaluation of the ability of Gal-NP@siBACE1 treatment to modulate AD hallmarks in APP/PS1 mice. (A) Mechanistic explanation for the effects
of siBACE1 therapy. (B) Representative Western blot data for BACE1 protein expression in hippocampus and cortex from nanocarrier-treated APP/PS1 mice, control APP/
PS1 groups, and WT mice. Quantification of Western blotting analysis of BACE1 expression was relative to -actin (n = 3, mean with SEM, *P < 0.05, **P < 0.01). (C) Representa-
tive confocal laser scanning microscopy imaging data assessing amyloid plaque burden. Immunofluorescence of A plaques (green) in hippocampus and cortex from
APP/PS1 transgenic and WT mice. Nuclei were stained by DAPI (blue). Scale bars, 100 m. (D) Percent surface area of amyloid plaques in hippocampus (left) and cortex
(right) regions was quantified. Data are presented as mean ± SEM; n = 4, **P < 0.01. (E) p-tau and (F) MBP expression in the hippocampus and cortex for nanocarrier-treated
APP/PS1 mice, control APP/PS1 groups, and WT mice (top). Quantification of Western blotting analysis was relative to -actin (bottom) (n = 3, mean with SEM, *P < 0.05,
**P < 0.01). All samples were collected after 10 administrations of nanomedicine.

Zhou et al., Sci. Adv. 2020; 6 : eabc7031 9 October 2020 7 of 14

SCIENCE ADVANCES | RESEARCH ARTICLE

A B

Levels in plasma (U/liter)

C Il-1 Il-6 Tnf-

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D
Il-1 Il-6 Tnf-

PBS
Gal-NP@siRNA

E Heart Liver Spleen Lung Kidney Cortex Hippocampus

WT-PBS
AD-PBS
NP@siBACE1
AD-Gal-

Fig. 6. Cytotoxicity and in vivo biocompatibility assessment of the Gal-NP@siRNA nanomedicine. (A and B) Blood chemistry examinations. Assessment of plasma
alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), plasma urea (BUN), creatinine (CR), and uric acid (UA) levels after a
single-dose nanomedicine treatment. n = 4, mean with SEM. (C and D) Core proinflammatory cytokines such as Il-1, Il-6, and Tnf- in liver (C) and kidney (D) were assessed
after a single-dose PBS or Gal-NP@siRNA nanomedicine treatment at days 2 and 14. n = 3, mean with SEM. (E) Representative data for hematoxylin and eosin staining in
major organs from APP/PS1 and control WT mice treated with Gal-NP@siBACE1 or PBS in the 10-time injection therapeutic experiments. Scale bars, 50 m.

Zhou et al., Sci. Adv. 2020; 6 : eabc7031 9 October 2020 8 of 14

SCIENCE ADVANCES | RESEARCH ARTICLE

within 2 weeks after injection, indicating that the Gal-NP@siRNA In addition to the stability challenge, effective BBB penetration
nanomedicine has a suitable safety profile and can survive free is another big challenge for nanotechnology-based brain disease
circulation in blood before its arrival at the target site in the brain. therapies. By design, our Gal-NP@siRNA–incorporated d-galactose
In addition, during nanomedicine therapy, there were no differences makes it possible to exploit glycemic Glut1 recycling, enhancing the
between treatment groups in body weight change or food uptake, delivery of Gal-NP@siRNA across the BBB upon glucose adminis-
further indicating the safety of the glycemia-controlled strategy for tration. Potentially, this BBB-penetrating strategy is feasible clinically,
Gal-NP@siBACE1 nanocarriers (fig. S9B). Histochemical staining as blood glucose spikes are easily induced through oral (glucose
showed that treatment with Gal-NP@siBACE1 nanocarriers induced drink) or intravenous administration. Glut1 transporter expression
no necrosis or apoptosis in major organs (Fig. 6E) after 10 cycles of and nutrition uptake decrease with age in AD patients (43, 44).
nanomedicine treatment. From this aspect, it may have an impact on the therapeutic efficiency
of Gal-NP@siRNA to some extent, especially in a senior AD model.
Another controversial point is that researchers found that BBB per-
DISCUSSION meability increased in AD, which contributes to cognitive decline
Integration of RNAi together with nanotechnology holds great independently of AD pathology (45, 46). However, the ineffective
promise for AD therapy. However, nanotechnology-based therapy AD therapeutic ability of nontargeted NP@siRNA signifies the
has been put off by stringent regulatory framework (12, 38). Clinical importance of Glut1-mediated transport for galactose-modified
translation of nanomedicines is often hindered by multiple factors, nanomedicines crossing the BBB.
including physical and chemical stability of nanomedicine, pharma- In addition to excellent blood stability and effective BBB pene-
cokinetic parameters, in vivo release mechanism, the robustness of tration, we also show that Gal-NP@siRNA nanomedicine exerts
manufacturing, route of administration, bioavailability, distribu- high brain accumulation. Gal-NP@siBACE1 decreased BACE1

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tion, biodegradation, safety, accumulation, and appropriate animal expression, leading to reduced levels of A plaques with the added
studies (39, 40). Most published studies examining nanomedicines benefit of suppressed phosphorylated tau protein levels and regener-
as potential AD therapeutics address a limited number of these ation of impaired myelin. These positive pathophysiological effects
factors, which probably explains why these have largely been unsatis- probably contributed to the restoration of cognitive performance of
factory in terms of efficacy and efficiency. In the work of Singer et al. Gal-NP@siBACE1–treated transgenic mice. In addition, Gal-NP@
(13), a siBACE1 lentiviral vector was applied by intracerebral injec- siRNA also exhibited excellent biocompatibility and did not cause
tions. This early proof-of-concept study showed considerable potential renal or hepatic responses or adverse effects on myelination, sug-
in BACE1 silencing strategy. However, several critical limitations gesting that there is an effective clearance of by-products from the
including toxicity, immunogenicity, and inflammatory response brain most likely via the paravascular glymphatic pathway (47).
hinder the therapeutic use of the lentiviral vector in CNS disease AD is a complex neurodegenerative disorder, and the pathological
(41). Also, intracerebral injection of the siRNA is destructive and pathways that govern AD are still controversial. Nonetheless, the
may cause multiple side effects. Exosomes hold excellent capacity toxic A accumulation and tauopathy are two of the most reliable
for reduce immunogenicity and siBACE1 brain delivery (14). How- pathologic events in AD progression. In several early studies, inhi-
ever, preparing enough quantity of purified exosomes remains bition of BACE1 mRNA levels lowers -secretase activity associated
challenging when this concept moves to clinics. Polymeric NPs are with BACE1 and consequently reduces production of APPs and
easier to synthesize and scale up and also display low immunogenicity. other proteins in cells (48–50). Therefore, BACE1 is considered one
A pioneering study developed synthesized polymeric nanoplatforms of the top drug targets for lowering cerebral A plaque levels in AD.
that deliver siRNAs to AD mice brain, but poor in vivo stability and We demonstrated partial knockdown of BACE1 protein expres-
ineffective brain accumulation limit its therapeutic effect (15). Still, sion, but there are several variants of that protein, and in subsequent
more powerful nanomaterials are urgently needed to overcome current studies, our Gal-NP@siRNA could be applied to carry multiple siRNAs
poor blood stability and inefficient BBB penetration (42). Here, we for holistic therapy. In addition, our therapeutic approach is suit-
developed a self-assembly method based on galactose-decorated able for “gene therapy cocktail” applications.
triple-interaction stabilized polymeric siRNA nanomedicine In summary, we developed an effective strategy to deliver siBACE1
(Gal-NP@siRNA). Our nanomedicine formulation produces siRNA-­ through the BBB with good circulation stability, which ameliorated
loaded NPs through simple mixing of prepared polymers with the AD-like pathology in APP/PS1 transgenic mice. These results indicate
required siRNA sequence in a tunable ratio manner. The simplicity that our Gal-NP@siRNA nanomedicine has good clinical translation
and versatility of our nanomedicine formulation can support assembly potential for AD therapy owing to ease of formulation, stability, and
of NPs loaded with any short nucleic acids. We demonstrate that BBB penetration. Furthermore, our Gal-NPs could also be used to
our nanomedicine formulation has superior physiological stability deliver siRNA in a wide range of CNS disease therapy including
and blood longevity that is critical for siRNA to achieve high accumu- other neurodegenerative conditions and brain cancer.
lation at diseased sites. To date, most reported siRNA nanomedicines
were solely electrostatically stabilized, which makes them susceptible
to dissociation in vivo, leading to short circulation time in blood. In MATERIALS AND METHODS
this study, the combination of Gu+/PO34− salt bridge that promotes Materials
additional electrostatic and hydrogen bond interaction with the See the Supplementary Materials for the synthesis of MeO-PEG-b-
fluorine-mediated hydrophobic interaction overcomes the stabili- P(GuF) and Gal-PEG-b-P(Gu). Primary antibody BACE1 (Abcam,
zation problem of typical siRNA nanomedicines and further endows AB183612), p-tau (Abcam, AB151559), MBP (Abcam, AB40390),
Gal-NP@siRNA with superior physiological stability and prolonged -actin (Abcam, AB8226), or glyceraldehyde-3-phosphate dehydro-
blood circulation time. genase (GAPDH) (Abcam, AB181602) and mouse or rabbit secondary

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SCIENCE ADVANCES | RESEARCH ARTICLE

antibody (LI-COR IRDye 800CW) were used. All siRNAs were syn- Flow cytometry assay
thesized by GenePharma Company, and the sequences used were as Neuro-2a cells were seeded in a six-well plate (1 × 106 cells per well)
follows: (i) Scramble: 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and incubated with PBS, Gal-NP@Cy5 siRNA, NP@Cy5-siRNA,
(sense) and 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (antisense); and naked Cy5-siRNA in 500-l medium (200 nM Cy5-siRNA) at
(ii) BACE1: 5′-GAACCUAUGCGAUGCGAAUdTdT-3′ (sense) and 37°C for 4 hours. The cells were digested by 0.25% (w/v) trypsin and
5′-AUUCGCAUCGCAUAGGUUCdTdT-3′ (antisense). The siBACE1 0.03% (w/v) EDTA. The suspensions were centrifuged at 1000 rpm
sequence was shown in a previous work, which showed a better for 3 min, washed twice with PBS, and then resuspended in 500 l
silencing effect among several sequences (51). A dye was introduced of PBS. Fluorescence histograms were immediately recorded with a
to the 5′-end of the antisense strand of siScr. For quantitative poly- BD FACSCalibur flow cytometer (Becton Dickinson, USA) and
merase chain reaction (qPCR), all of these primers are designed by analyzed using CellQuest software based on 10,000 gated events.
Primer-BLAST (National Center for Biotechnology Information) and The gate was arbitrarily set for the detection of Cy5 fluorescence.
listed in table S1.
In vitro cytotoxicity assay
Nanocarrier characterization Neuro-2a, SH-SY5Y, and PC-12 cells were seeded in 96-well plates
1
H nuclear magnetic resonance spectra were recorded on AVANCE (6000 cells per well) and incubated in 100 l of culture medium for
III HD 400 MHz (Bruker, Switzerland). The size was determined at 24 hours. Thereafter, PBS, NP@siRNA, Gal-NP@siRNA, and naked
25°C using DLS (Zetasizer Nano-ZS, Malvern Instruments) equipped siRNA NPs were added to the cells, and the cells were then incubated
with a 633-nm He-Ne laser using backscattering detection. TEM for 48 hours. At assay end, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-
was performed using a JEM-2100 TEM operated at an accelerating phenytetrazoliumromide (MTT) solution (5 mg/ml, 1 l/10 l of
voltage of 200 kV (JEOL, Japan). The confocal laser scanning micros- medium) was added and samples were further incubated at 37°C for

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copy images of cells were taken on a Zeiss Confocal Microscope 4 hours. Cell viability was determined from the absorbance of extra-
system (Zeiss 880). The transfected cells were observed with a cellular medium at 570 nm.
microplate reader (Molecular Devices, USA), and fluorescence was
quantitatively measured by flow cytometry (BD FACSCalibur, Gene silencing assay by quantitative real-time PCR
San Jose). The gel electrophoresis images were taken by Molecular Endogenous BACE1 gene silencing activity of Gal-NP@siRNA was
Imager FX (Bio-Rad, Hercules, CA). The fluorescent images were investigated by quantitative real-time PCR (qRT-PCR). Neuro-2a
scanned using a near-infrared fluorescence imaging system (Lumina, cells were seeded in a six-well plate (1 × 106 cells per well) in growth
IVIS III). medium (Minimum Essential Medium/Earle’s Balanced Salt Solu-
tion also known for MEM/EBSS containing 10% FBS) for 24 hours.
Gel retardation assay The medium was removed and replenished with fresh medium
siRNA (0.5 mg) was dissolved in 500 l of diethyl pyrocarbonate– (1000 l) containing PBS, NP@siScr, NP@siBACE1, Gal-NP@siScr,
treated water. Polymer and siRNA solutions were mixed (the amount and Gal-NP@siBACE1 (400 nM siRNA). After 3 days, the cells were
of siRNA was 2 M) at polymer/siRNA weight ratios of 1:1, 2.5:1, washed with PBS and the total RNA was extracted using the RNeasy
5:1, 10:1, 15:1, and 20:1. The mixture was incubated for 30 min. The Mini Kit (Qiagen).
siRNA binding ability of polymer was studied by agarose gel. The For mice experiments, normal female Balb/c mice were randomly
polymer/siRNA ratios were electrophoresed through a 2% agarose divided into two treatment groups (n = 3). PBS and Gal-NP@siScr
gel containing Gel Red at 35 V in TAE solution [40 mM tris-HCl, (1 mg of siRNA equiv./kg) were intravenously injected into mice via
1% acetic acid (v/v), and 1 mM EDTA]. Fluorine-free Gal-NP the tail vein (n = 3 per group). At prescribed time points after injec-
(mixed with siRNA at a mass ratio of 10:1) and fluorinated Gal-NP tion, animals were anesthetized and transcardially perfused with
(mixed with siRNA at a mass ratio of 2.5:1) were prepared, and saline. The tissues were homogenized in 1 ml of ice-cold TRIzol
then different doses of heparin solution were added to the NPs. reagent (Invitrogen) according to the protocol of the manufacturer.
siRNA release was measured by a nucleic acid gel at different Reverse transcription and qPCR were carried out by following
time points. reverse transcription protocol (Takara) and SYBR Green Gene Ex-
pression Assays Protocol (Takara) with the Roche LightCycler 480
siRNA nanomedicine formulation RT-PCR System. mGAPDH was used as an endogenous house-
The siRNAs were dissolved in Hepes buffer (pH 7.4) to a stock con- keeping gene to normalize the Bace1 mRNA. The mRNA expres-
centration of 4000 nM (53.2 g/ml) for in vitro experiments and sion level was calculated based on comparative Ct method (2−∆∆Ct).
400 g/ml for animal experiments. The polymer solution was added
into the siRNA solution (volume ratio = 1:1) and then gently pipetted Animals
10 times and incubated for 30 min at room temperature. Then, the All animals used for experiments were allocated blindly to treatment
prepared nanomedicines were diluted into a working concentration groups. All protocols were approved by the Animal Care and Use
of 200 or 400 nM siRNA for in vitro experiments and 200 g/ml for Committee of Laboratory Animal Center, Henan University (ethics
in vivo experiments. Gal-PEG-b-P(Gu) and MeO-PEG-b-P(Gu)/ approval number: HUSOM-2018-354). Adult, 8-week female Balb/c
MeO-PEG-b-P(GuF) (molar ratio = 1:3) complexed with siRNA mice were used in the biodistribution assay, in vivo BACE1 silenc-
yielded nanomedicine denoted as fluorine-free Gal-NP@siRNA ing, and blood biochemistry examinations. For in vivo imaging
(polymer/siRNA weight ratio = 10) and NP@siRNA (polymer/ experiments, 8-week female nude mice were used. For therapeutic
siRNA weight ratio = 2.5:1). Gal-PEG-b-P(Gu)/MeO-PEG-b-P(GuF) evaluation experiments and animal behavior tests, 8-month male
(molar ratio = 1:3) complexed with siRNA yielded Gal-NP@siRNA APP/PS1 and C57BL/6 mice (WT-like littermates) were used. All
nanomedicine (polymer/siRNA weight ratio = 2.5:1). mice were provided by Beijing Vital River Laboratory Animal

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SCIENCE ADVANCES | RESEARCH ARTICLE

Technology Co. Ltd. and transported to the animal facility at Nankai To evaluate the ex vivo brain targeting and NP biodistribution,
University or Henan University at least 2 weeks before testing. Mice Gal-NP@Cy5-siRNA and NP@Cy5-siRNA were injected intravenously
were housed in a standard individual ventilation cages animal exper- to nude mice after fasting. One hour after injection, main organs
imental system (Suzhou Fengshi Laboratory Animal Equipment Co. were separated, washed in PBS, and monitored.
Ltd.) with corn cob bedding and a wire lid, providing climbing oppor-
tunities (APP/PS1 and its control mice were housed one mouse per cage Western blot
for experiments). Mice were kept under a 12:12-hour day-night Neuro-2a cells were harvested at day 3 after incubation with Gal-
schedule; food and water were available ad libitum. Separate cohorts NP@Cy5-siRNA and control vectors. For sufficient siBACE1 verifi-
were used for each experiment (n = 6 to 8 per treatment group, WT- cation, mouse brain tissues were taken from Balb/c mice after two
PBS = 8, AD-PBS = 7, AD-NP@siBACE1 = 6, AD-Gal-NP@siScr = 6, siRNA injections. In therapeutic evaluation, mouse brain tissues were
AD-Gal-NP@siBACE1 = 7). Food was removed for fasting 24 hours taken 1 day after completion of behavioral assessments. Animals
before nanomedicine treatment. Two hundred microliters of 20 weight were anesthetized and transcardially perfused with saline. Tissue
% (wt %) glucose was administered to all groups by intraperitoneal (whole hippocampus and cortex) and cells were homogenized in
injection 30 min before nanomedicine injection. For nanomedicine radioimmunoprecipitation assay lysis buffer with a proteinase and
treatment, we administered NPs modified with or without galactose phosphorylase inhibitor cocktail (Thermo Fisher Scientific) and
(Gal-NP@siBACE1 or NP@siBACE1) to verify Glut1 targeting centrifuged for 15 min (12,000 rpm, 4°C). The protein concentra-
and effective brain delivery. Gal-NPs loaded with siBACE1 or siScr tion of the supernatant was determined using the BCA Protein
(Gal-NP@siBACE1 or Gal-NP@siScr) were designed to assess Assay Kit (Beyotime, China). Standard Western blot electrophoresis
gene silencing efficiency. APP/PS1 and WT-like mice injected with was then performed, with proteins transferred onto polyvinylidene
200 l of PBS were used as controls to demonstrate pathological difluoride membranes (Millipore 0.22 m) and immunoblotted.

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dysfunction in AD mice. All AD nanomedicine therapy groups Primary antibody BACE1 (Abcam, AB183612), p-tau (Abcam,
were given 1 mg of siRNA equiv./kg diluted in 200 l of PBS via AB151559), MBP (Abcam, AB40390), -actin (Abcam, AB8226), or
caudal vein injection every 3 days. Treatment schedules for injec- GAPDH (Abcam, AB181602) and mouse or rabbit secondary anti-
tion of siRNA nanomedicines and behavior test date are highlighted body (LI-COR IRDye 800CW) were used. Data quantification was
in Fig. 4A. performed by ImageJ software.

Pharmacokinetics Confocal microscopy imaging

Gal-NP@Cy5-siRNA, fluorine-free Gal-NP@Cy5-siRNA, and naked For cellular uptake assay, Neuro-2a cells were cultured on micro-
Cy5-siRNA (1 mg of Cy5-siRNA equiv./kg) in 200 l of Hepes were scope slides in 24-well plates (1 × 105 cells per well) and incubated
intravenously injected into mice via the tail vein (n = 3). At pre- with NP@FAM-siRNA, Gal-NP@FAM-siRNA, or naked FAM-siRNA
scribed time points after injection, ~50 l of blood was taken out in 500 l of medium (200 nM FAM-siRNA) at 37°C. The culture
from the eye socket of mice. The blood samples were immediately medium was removed, and the cells were washed three times with
dissolved in 0.6 ml of lysis buffer (1% Triton X-100) at 37°C over- PBS, fixed with 4% paraformaldehyde solution for 15 min, and
night followed by centrifugation (14,000 rpm, 30 min). The Cy5 washed three times with PBS. Cells were then permeabilized with
level in the supernatant was determined by fluorometry. The blood 0.1% Triton X-100 in PBS for 15 min and then stained with a tetra­
circulation followed a typical two-compartment model: a rapid de- methyl rhodamine isothiocyanate (TRITC) fluorescent phalloidin
cline in the distribution phase and a long period in the elimination conjugate solution (10 g/ml) in PBS (containing 1% dimethyl sulf-
phase. We calculated the half-lives of two phases (t1/2, and t1/2,) by oxide from the original stock solution) for 30 min at room tempera-
fitting the experimental data using SoftwareS6 Origin 8 exponential ture and washed three times with PBS. The cell nuclei were stained
decay 2 model: y = A1 × exp(−x/t1) + A2 × exp(−x/t2) + y0, and then with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and washed
taking t1/2, = 0.693 × t1 and t1/2, = 0.693 × t2. three times with PBS. The fluorescence images were obtained using
a confocal microscope (Zeiss 880).
Biodistribution For endosomal escape, Neuro-2a cells were cultured on micro-
After fasting (1 day), glucose solution (20 wt %) was injected to elevate scope slides in 24-well plates (1 × 105 cells per well) and incubated
the blood glucose concentration, and 30 min later, a single dose of with Gal-NP@FAM-siRNA in 500 l of medium (200 nM FAM-siRNA)
NP@Cy5-siRNA and Gal-NP@Cy5-siRNA in 200 l of Hepes was at 37°C for 2, 4, 6, and 8 hours. At the determined time, the culture
administrated intravenously via the tail vein (1 mg of siRNA equiv./kg). medium was removed and the cells were washed three times with
After 1 hour, the mice were sacrificed. The major organs including PBS and incubated with LysoTracker (50 nM, Invitrogen) at 37°C
heart, liver, spleen, lung, kidney, and brain were collected, washed, for 30 min and then with Hoechst 33342 (Solarbio, 10 g/ml) for
dried, weighed, and homogenized in 0.6 ml of 1% Triton X-100 with 10 min to visualize the endosomes/lysosomes and nuclei. Thereafter,
a homogenizer at 14,000 rpm for 30 min. Cy5 in the supernatant cells were washed three times with PBS and fixed with 4% para-
was determined by fluorometry based on a calibration curve and formaldehyde solution for 15 min. Fluorescence images were obtained
expressed as injected dose per gram of tissue (%ID/g). using a confocal microscope (Zeiss 880).
For tissue immunofluorescence, brains were fixed in 4% para-
In vivo and ex vivo imaging formaldehyde for 24 hours, then dehydrated, embedded, and cut
To evaluate the in vivo brain targeting ability of NPs, Gal-NP@ into 8-m frozen slices. The sections were washed three times with
Cy5-siRNA and NP@Cy5-siRNA were injected intravenously to PBS, blocked with normal goat serum for 1 hour, and subsequently
nude mice and monitored at different time points by using the Lumina incubated with anti–-amyloid primary antibody (1:200, BioLegend,
IVIS III Imaging System (excitation = 620 nm; emission = 670 nm). catalog no.803001) or anti-BACE1 primary antibody (1:250, Abcam,

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SCIENCE ADVANCES | RESEARCH ARTICLE

AB183612) overnight at 4°C. Sections were then washed three times 2 points, paper towels are concentrated in the cage, but no obvious
with PBS, and the slices were incubated with fluorescein isothiocyanate bite marks; 3 points, the paper towel was concentrated on one side
(FITC)–conjugated goat anti-mouse immunoglobulin G (IgG) or one corner with some bite marks; 4 points, most of the paper
(1:200, Abcam) or FITC-conjugated goat anti-rabbit IgG (1:200, towels were bitten and gathered together. The paper, which was
Jackson) for 1  hour at room temperature. Then, the slices were shredded into small pieces or full of holes, was defined as bitten. All
stained with DAPI (10 g/ml) for 10 min. The fluorescence images results were scored blindly.
were obtained using a confocal microscope (Zeiss 880), and fluores-
cence intensity was analyzed by ImageJ software. Morris water maze
To evaluate spatial learning and memory, MWM was performed in
Blood biochemistry and blood routine examinations accordance with standard protocols (52). The pool was divided into
Healthy Balb/c female mice at age 6 to 8 weeks were randomly four quadrants (Fig. 4G), and on the wall of each quadrant, a differ-
divided into two treatment groups (n = 3). PBS and Gal-NP@siScr ent symbol (pentagram, square, triangle, and circle) was affixed to
(1 mg of siRNA equiv./kg) were intravenously injected into mice via provide extra-maze spatial cues. The water temperature was kept at
the tail vein (n = 3 per group). At prescribed time points after injec- 22 ± 1°C, and highly dispersed food-grade titanium dioxide was
tion, blood was collected via eye socket bleeding. For blood bio- added into water to aid animal tracking. All MWM experiments
chemistry examination, whole blood was centrifuged at 800g for were carried out daily and, at the same time, in the afternoon. The
5  min to collect serum for analysis. Standard blood chemistry experimental equipment was kept in a confined space without noise
parameters were analyzed using a kit from Wuhan Servicebio Tech- or strong light sources.
nology Co. Ltd. on an automated chemistry analyzer (Chemray 240 Mice were habituated to the room for 2 hours before the experi-
Rayto lnc.). Blood cell parameters were analyzed with an automated ment. Each mouse was trained to find a hidden platform for five

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blood cell analyzer (BC-2800Vet-Mindray Inc.). consecutive days with four trials per day, with a 20- to 30-min inter-
trial interval. The mice were put into the water with their heads
Novel object recognition facing the wall of the pool, and the platform was allocated random-
The NOR test was performed according to published methods (32). ly to one of the four quadrants. The time the animals took to find
The experimental apparatus was a polyethylene white rectangular the platform was recorded. In the training sessions, if the latency to
open field box (50 cm by 50 cm by 50 cm). Habituation took place find the platform exceeded 60 s, the animals were guided to the
by exposing the animal to the experimental apparatus for 10 min in platform and kept there for 10 s. Mice were trained for 5 days to
the absence of objects on the day before training. During the train- find the platform. Twenty-four hours after training, the platform
ing phase, mice were placed in the experimental apparatus in the was removed and the 60-s probe test commenced. Animals were
presence of two identical objects (odorless wood cuboid or pyramid placed into the water facing the quadrant, which was opposite the
was used to prevent mice from climbing onto the object, avoid mice target quadrant. The time spent in the target quadrant and number
preference and sitting on it) and were allowed to explore the object of crossing the platform location was recorded as an indicator of
for 10 min. After 24 hours, mice were placed again in the apparatus, spatial memory.
where this time one of the objects was replaced by a novel one. Mice
were allowed to explore for 10 min. DI and PI were used to assess Statistical analysis
NOR; this index accounts for differences in exploration time. DI Results were analyzed by using GraphPad Prism software. Differ-
and PI are calculated as the time spent exploring (total exploration ences between two groups were assessed using unpaired t tests. For
of at least 30 s, sniffing, trying to move, and front paw pushing the multiple comparisons, statistical significance was analyzed using
objects were defined as exploring, but not the time spent near the one-way analysis of variance (ANOVA), followed by Fisher’s least
objects without investigation, or passing by the objects). Data were significant difference post hoc test, which was used when compar-
collected using tracking software, and manual scoring was used to ing all the conditions. Statistical differences in behavioral data were
assess behaviors from the videos. DI was calculated as the time determined using two-way repeated-measures ANOVA. The level
spent exploring the novel object minus the time spent exploring the of statistical significance was set at P < 0.05. *P < 0.05 was consid-
familiar object, divided by the total exploration time. PI was calcu- ered significant, and **P < 0.01 ***P < 0.001 were considered highly
lated as the proportion of total time spent exploring new or old ob- significant. All data were expressed as mean ± SEM unless otherwise
ject. [DI = Tnovel − Tfamiliar/(Tnovel + Tfamiliar), PI = Tnovel or Tfamiliar/ indicated.
(Tnovel + Tfamiliar)]. All DI values fall between −1 and +1, and PI val-
ues fall between 0 and 1.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
Nest construction content/full/6/41/eabc7031/DC1
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52. P. Zhao, X. Qian, Y. Nie, N. Sun, Z. Wang, J. Wu, C. Wei, R. Ma, Z. Wang, G. Chai, analyzed the data. M.Z. and F.Z. led the design, preparation, and functional characterization of
Neuropeptide S ameliorates cognitive impairment of APP/PS1 transgenic mice by nanocomplexes. X.X. and Y.Zh. led the animal experiments. M.Z. and Y.L. wrote the manuscript
promoting synaptic plasticity and reducing A deposition. Front. Behav. Neurosci. 13, 138 and revised it according to the comments of Y.A., O.S., A.I.B., J.L., X.P., W.T., Y.Zo., and B.S. All
(2019). authors participated in the discussion of the project. Competing interests: The authors declare
that they have no competing interests. Data and materials availability: All data needed to
evaluate the conclusions in the paper are present in the paper and/or the Supplementary
Acknowledgments: We thank K. Kataoka at University of Tokyo and R. Chung at Macquarie Materials. Additional data related to this paper may be requested from the authors.
University for scientific discussions and suggestions. Funding: This work was supported by the
National Natural Science Foundation of China (NSFC U1804139, U1604177, 31800841, Submitted 13 May 2020
31922045, 31771031, and 81701829), National Key Technologies R&D Program of China Accepted 26 August 2020
(2018YFA0209800), Key Research Program in Colleges and Universities of Henan Province Published 9 October 2020
(19zx006), Australian Endeavour Fellowship (no. 69172018), Mason Foundation National 10.1126/sciadv.abc7031
Medical Program (MAS2017F034), National Health and Medical Research Council (NHMRC)
Dementia Fellowship (GNT1111611), and NHMRC Project Grant (GNT1166024). Author Citation: Y. Zhou, F. Zhu, Y. Liu, M. Zheng, Y. Wang, D. Zhang, Y. Anraku, Y. Zou, J. Li, H. Wu,
contributions: M.Z., X.X., and B.S. designed the concept, supervised the project, and revised X. Pang, W. Tao, O. Shimoni, A. I. Bush, X. Xue, B. Shi, Blood-brain barrier–penetrating siRNA
the manuscript. F.Z., Y.Zh., Y.L., Y.W., D.Z., Y.Zo., and H.W. performed the experiments and nanomedicine for Alzheimer’s disease therapy. Sci. Adv. 6, eabc7031 (2020).

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Zhou et al., Sci. Adv. 2020; 6 : eabc7031 9 October 2020 14 of 14

 Blood-brain barrier–penetrating siRNA nanomedicine for Alzheimer’s disease
therapy
Yutong Zhou, Feiyan Zhu, Yang Liu, Meng Zheng, Yibin Wang, Dongya Zhang, Yasutaka Anraku, Yan Zou, Jia Li, Haigang
Wu, Xiaobin Pang, Wei Tao, Olga Shimoni, Ashley I. Bush, Xue Xue, and Bingyang Shi

Sci. Adv., 6 (41), eabc7031.

DOI: 10.1126/sciadv.abc7031

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