a targeting nanotherapy for abdominal aortic aneurysms · 2018-11-14 · a targeting nanotherapy...

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J O U R N A L O F T H E AM E R I C A N C O L L E G E O F C A R D I O L O G Y V O L . 7 2 , N O . 2 1 , 2 0 1 8

ª 2 0 1 8 B Y T H E AM E R I C A N C O L L E G E O F C A R D I O L O G Y F O UN DA T I O N

P U B L I S H E D B Y E L S E V I E R

A Targeting Nanotherapy forAbdominal Aortic Aneurysms
Juan Cheng, BSC,a Runjun Zhang, MS,a,b Chenwen Li, MS,a Hui Tao, BSC,a Yin Dou, PHD,a Yuquan Wang, MS,a,b
Houyuan Hu, MD,b Jianxiang Zhang, PHDa

ABSTRACT

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BACKGROUND Abdominal aortic aneurysm (AAA) is a leading cause of mortality and morbidity in the elderly.

Currently, there remain no effective drugs that can prevent the growth of aneurysms and delay aneurysm rupture in the

clinical setting.

OBJECTIVES The aim of this study was to develop a nanotherapy that can target aneurysms and release drug mole-

cules in response to the inflammatory microenvironment.

METHODS Using a reactive oxygen species (ROS)-responsive nanoparticle and a candidate drug rapamycin, in combi-

nation with a peptide ligand for integrin and biomimetic cloaking with macrophage cell membrane, a nanotherapy was

developed. Its effectiveness was demonstrated by in vitro and in vivo studies.

RESULTS Based on a facile and translational method, a rapamycin-loaded responsive nanotherapy was successfully

prepared, which could release drug molecules upon triggering by the high level of ROS. In cells associated with the

development of AAAs, the nanotherapy significantly inhibited calcification and attenuated ROS-mediated oxidative stress

and apoptosis. By passively targeting aneurysms and releasing drug molecules in response to the inflammatory micro-

environment, the intravenously injected ROS-responsive nanotherapy more effectively prevented aneurysm expansion in

AAA rats than a nonresponsive control nanotherapy. After decoration with a peptide ligand cRGDfK and macrophage cell

membrane, the aneurysmal targeting capability and therapeutic effects of a ROS-responsive nanotherapy with a mean

diameter of 190 nm were further enhanced. Moreover, the nanotherapy showed a good safety profile in a preliminary

safety test.

CONCLUSIONS The multifunctional nanotherapy can be further studied as a promising targeted drug for treatment of

aneurysms. The underlying design principles enable the development of a broad range of nanomedicines for targeted therapy

of other vascular diseases. (J Am Coll Cardiol 2018;72:2591–605) © 2018 by the American College of Cardiology Foundation.

A bdominal aortic aneurysm (AAA) is a leadingcause of mortality and morbidity in theelderly (1). Pathologically, infiltration of large

numbers of inflammatory cells (2), elevated levels ofmatrix metalloproteinases (MMPs) (3), overproducedreactive oxygen species (ROS) (4), intimal and medialcalcification (5), neovascularization (6), apoptosis ofvascular smooth muscle cells (VSMCs) (7), and degra-dation of elastic lamellae aorta (3) are involved in an-eurysms. Despite no symptoms before rupture,

N 0735-1097/$36.00

m the aDepartment of Pharmaceutics, College of Pharmacy, Third Milit

epartment of Cardiology, Southwest Hospital, Third Military Medical Univ

the National Natural Science Foundation of China (Nos. 81471774 & 817018

uthwest Hospital (No. SWH2016ZDCX1016), and the Science and Tech

Southwest Hospital (No. SWH2016LHYS-05). The authors have reported tha

this paper to disclose.

nuscript received January 22, 2018; revised manuscript received August

rupture of AAAs is often lethal, with mortality of85% to 90% (8).

Mechanical intervention, including open andendovascular repair, is currently the only effectivetreatment that can prevent aneurysm-related death(9). Unfortunately, open surgery is associated withperioperative mortality and morbidity (10). On theother hand, approximately 20% to 30% of patientswho undergo endovascular repair require reinter-vention within 5 years due to reperfusion and the

https://doi.org/10.1016/j.jacc.2018.08.2188

ary Medical University, Chongqing, China; and the

ersity, Chongqing, China. This study was supported

32), the Innovation Program for Key Technologies of

nology Innovation Program in Military Medicine

t they have no relationships relevant to the contents

14, 2018, accepted August 20, 2018.


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ABBR EV I A T I ON S

AND ACRONYMS

AAA = abdominal aortic

aneurysm

Ca/Pi = calcium and inorganic

phosphorus

CMV = cell membrane vesicle

CROR NP = a reactive oxygen

species–responsive, cRGDfK

targeted, and macrophage cell

membrane-coated rapamycin

nanotherapy

DHE = dihydroethidium

IFN = interferon

IL = interleukin

IV = intravenous

MCP = monocyte

chemoattractant protein

MMP = matrix

metalloproteinase

NP = nanoparticle

OCy5 NP = Cy5-labeled OxbCD

nanoparticle

OR NP = reactive oxygen

species–responsive rapamycin

nanotherapy

OxbCD = oxidation-responsive

b-cyclodextrin material

PEG = polyethylene glycol

PR NP = PLGA-derived

rapamycin nanotherapy

RAP = rapamycin

ROR NP = reactive oxygen

species–responsive, cRGDfK

targeted rapamycin

nanotherapy

ROS = reactive oxygen species

TNF = tumor necrosis factor

VSMC = vascular smooth

muscle cell

Cheng et al. J A C C V O L . 7 2 , N O . 2 1 , 2 0 1 8

A Targeting Nanotherapy for Abdominal Aortic Aneurysms N O V E M B E R 2 7 , 2 0 1 8 : 2 5 9 1 – 6 0 5

2592

associated risk of aneurysm rupture (11). Inaddition, for small aneurysms and aneu-rysms with anatomic constraints, both repairmethods are not optimal strategies (8).Consequently, effective drugs that candecrease AAA expansion and prevent AAAsfrom rupture are imperative. Recent studiesindicated that synthetic inhibitors of MMPs(12) and angiotensin-converting enzyme (13)can suppress the expansion of experimentalAAAs. Rapamycin (RAP), an immunosup-pressant and an inhibitor of mammaliantarget of rapamycin, also prevented aneu-rysm growth in rats (14). Regardless ofpromising results in preclinical studies, noneof the existing drugs have provided benefi-cial effects in clinical trials (15). Additionally,administration of these drugs is generallyassociated with adverse effects due to theirnonspecific distribution. Also, poor watersolubility limits their clinical applications.Therefore, innovative strategies that canreduce side effects of antianeurysmal drugswhile maintaining or even potentiating theirefficacies are urgently needed to promoteclinical translation.

As is well documented, oxidative stress,resulting from overproduced ROS, is closelyassociated with AAA formation (4). On thebasis of this pathophysiological feature, wedeveloped a multifunctional nanotherapy fortargeted treatment of AAAs. By ligand andmacrophage cell membrane–mediated syn-ergistic targeting, the new nanotherapy caneffectively accumulate in AAAs and releasedrug molecules in response to high ROS inaneurysms.

SEE PAGE 2606

METHODS

Details of the materials and methods are available inthe Online Appendix.

SYNTHESIS OF A ROS-RESPONSIVE MATERIAL AND

PREPARATION OF DIFFERENT NANOPARTICLES.

A ROS-responsive material (oxidation-responsiveb-cyclodextrin material [OxbCD]) was synthesized bycovalently conjugating 4-(hydroxymethyl)phenyl-boronic acid pinacol ester (PBAP) onto b-cyclodextrin(16). A modified nanoprecipitation/self-assemblymethod was used to prepare different nanoparticles(NPs) (16). To prepare a ROS-responsive nanotherapycoated with macrophage cell membrane, cell mem-brane vesicles (CMVs) derived from macrophages

were first prepared (17), which were then coated ontothe cores of NPs by coextruding through a 200-nmpolycarbonate membrane.

IN VITRO HYDROLYSIS AND RELEASE TESTS.

In vitro hydrolysis of different NPs was conducted inphosphate-buffered saline with or without 1.0 mmol/lH2O2. The hydrolysis degree was calculated byquantifying the transmittance values at 500 nm atpredetermined time points. For in vitro release ex-periments, freshly prepared RAP nanotherapies wereplaced into dialysis tubing. At predetermined timeintervals, the RAP concentration in the release me-dium was quantified.

INTRACELLULAR UPTAKE STUDIES. RAW264.7mouse macrophages or VSMCs were seeded ontocoverslips in a 12-well plate. After incubation withCy5-labeled NPs for pre-defined time periods andstaining with 4,6-diamidino-2-phenylindole (DAPI),fluorescence images were acquired. In separate ex-periments, VSMCs and RAW264.7 cells were seeded ina 12-well plate and incubated with Cy5-labeled NPs.After 1, 2, or 8 h of incubation, fluorescence in-tensities in cells were analyzed by flow cytometry.

DETECTION OF CELL CALCIFICATION IN VSMCs.

VSMCs were seeded in a 24-well plate. After pre-treatment with different formulations for 24 h, cellswere induced with calcium and inorganic phosphorus(Ca/Pi) for 24 h. The cell calcification degree wasassessed by Alizarin Red staining. Besides directobservation, the calcium content and cell viability ofVSMCs were quantified.

QUANTIFICATION OF INTRACELLULAR ROS GENERATION

IN VSMCs. The intracellular ROS levels in VSMCsinduced by H2O2 were evaluated by dihydroethidium(DHE) staining. Cells were seeded onto coverslips in a12-well plate. After 24 h of incubation with differentformulations, cells were exposed to 400 mmol/l H2O2

for another 6 h. Fluorescence microscopy was per-formed after the nuclei were stained with DAPI. Inseparate studies, the intracellular ROS levels inVSMCs were quantified by flow cytometry.

IN VITRO APOPTOSIS ASSAY OF VSMCS. VSMCswere seeded in a 12-well plate. After 12 h of incuba-tion with various formulations, cells were treatedwith 400 mmol/l H2O2 for 6 h. Subsequently, apoptosisanalysis was performed by flow cytometry using aFITC Annexin V Apoptosis Detection Kit with 7-AAD(BioLegend, San Diego, California). Cell viability af-ter different treatments was also quantified.

ESTABLISHMENT OF AN EXPERIMENTAL AAA

MODEL IN RATS. Male Sprague-Dawley rats wereobtained from the Animal Center at the Third MilitaryMedical University (Chongqing, China). All animal


J A C C V O L . 7 2 , N O . 2 1 , 2 0 1 8 Cheng et al.N O V E M B E R 2 7 , 2 0 1 8 : 2 5 9 1 – 6 0 5 A Targeting Nanotherapy for Abdominal Aortic Aneurysms

2593

care and experiments were performed in accordancewith the Guide for the Care and Use of LaboratoryAnimals proposed by the National Institutes ofHealth. Local elastin damage in the abdominal aorticregion in rats was induced by perivascular applicationof calcium chloride (CaCl2). In all cases for the imag-ing and therapeutic studies, rats in the normal controlor AAA- group were surgically treated with saline,following the similar procedures.

IN VIVO TARGETING CAPABILITY OF DIFFERENT

NANOPARTICLES. Two weeks after AAA in rats wasinduced by CaCl2 infiltration, Cy7.5-labeled NPs wereadministered in randomly assigned rats via intrave-nous (IV) injection. At 8 h after different treatments,the thoracic aorta, abdominal aorta, and bilateral iliacarteries were harvested together for ex vivo imaginganalyses. In separate studies, the distribution ofnanoparticles in aneurysms was analyzed by immu-nofluorescence, after AAA rats were treated withdifferent Cy5-labeled NPs.

THERAPEUTIC EFFECTS OF NANOTHERAPIES IN

AAA RATS. AAA in rats was induced as aforemen-tioned. At day 2 or 10 after injury, nanotherapies wereIV injected in randomly assigned rats twice a week for3 weeks at 1.0 mg/kg of RAP. After treatment, theabdominal aortas were isolated for different qualita-tive and quantitative analyses.

STATISTICAL ANALYSIS. Statistical analysis wasperformed by SPSS 13.0 (IBM, Armonk, New York).Analyses of cellular uptake data were performed us-ing repeated measures analysis of variance in 2-wayanalysis with the least significant difference posthoc test, and the Mauchly’s test of sphericity wasused to evaluate heterogeneity of variances. Statisti-cal analyses in other cases were conducted using1-way analysis of variance with least significant dif-ference post hoc tests. p < 0.05 was considered to bestatistically significant.

RESULTS

DESIGN OF AN ADVANCED NANOTHERAPY. Fortargeted therapy of AAAs, we designed a ROS-responsive, integrin targeting, and macrophage-mimetic nanoplatform (Online Figure 1). The core ofthis nanoplatform consisted of an oxidation-responsive material, which is encased within a lipidshell anchored with polyethylene glycol (PEG) chainsto provide water solubility and long blood circulation.To enhance aneurysmal targeting, the shell of thisnanoplatform is further functionalized with a peptideligand and macrophage cell membrane (OnlineFigure 1A). After targeting to aneurysmal sites, this

nanoplatform can release therapeutic molecules upontriggering by ROS, thereby affording the desirableefficacy (Online Figure 1B).

DEVELOPMENT OF DIFFERENT RAP NANOTHERAPIES.

For the synthesized ROS-responsive material, eachOxbCD contained approximately 6 PBAP units (OnlineFigure 2A). Poly(D,L-lactide-co-glycolide) (PLGA), aU.S. Food and Drug Administration–approved biode-gradable polymer, was used as a nonresponsive ma-terial. OxbCD and PLGA NPs containing RAP wereprepared by nanoprecipitation (Figure 1A) (18), whichwere defined as OR NP and PR NP, respectively. Theobtained nanotherapies displayed a spherical shapeas observed by transmission electron microscopy(Figures 1B and 1C), with narrow size distribution.Both nanotherapies showed negative zeta-potential(Figure 1D). The average diameter was 197 � 3 mmand 167 � 4 nm, whereas the RAP content was 5.4 wt%and 7.8 wt% for PR NP and OR NP, respectively.

IN VITRO DRUG RELEASE OF NANOTHERAPIES. ForPR NP, <50% of total RAP was released from PR NPafter 72 h in phosphate-buffered saline with 0 or1.0 mmol/l H2O2 (Figure 1E). By contrast, RAP releasefrom OR NP was dramatically accelerated by1.0 mmol/l H2O2. Nearly complete RAP release(w96%) from OR NP was found at 72 h, significantlyhigher than that without H2O2. This result agrees withthe hydrolysis behaviors of corresponding blank NPs(Online Figure 2B). These in vitro tests demonstratedROS-responsive release characteristics of OR NP.

CELLULAR UPTAKE OF VARIOUS NANOPARTICLES.

Using Cy5-labeled NPs derived from PLGA (PCy5 NP)or OxbCD (OCy5 NP) (Online Figures 2C and 2D), weexamined cellular uptake of NPs in rat VSMCs andRAW264.7 murine macrophages, since both VSMCsand macrophages are involved in the pathogenesis ofAAAs (2). Fluorescence observation showed that PCy5NP and OCy5 NP were internalized by VSMCs andmacrophages in a time-dependent manner (Figures 1Fand 1G). Even at 1 h, considerable fluorescent signalsin both cells were observed. Flow cytometric quanti-fication demonstrated remarkably enhanced intra-cellular accumulation of NPs with prolongedincubation (Figures 1H and 1I). Of note, both PLGA andOxbCD NPs exhibited low cytotoxicity in VSMCs(Online Figure 2E).

NANOTHERAPIES INHIBIT CA/PI-INDUCED

CALCIFICATION AND CELL DEATH IN VSMCs.

Vascular calcification has been considered as a riskfactor for rupture of AAAs (5). We evaluated in vitroanticalcification effects of nanotherapies in ratVSMCs. After exposure to Ca/Pi, Alizarin Red stainingshowed significant calcium deposition in VSMCs of










FIGURE 1 Fabrication, Characterization, and Cellular Uptake of Different NPs

A

NanoprecipitationSelf-assembly

PR NP

PLGA

RAP

DSPE-PEG

OxbCD

OR NP

NanoprecipitationSelf-assembly

B

200 nm

PR NP

15

10

5

0103102

Diameter (nm)101 104

20

Inte

nsity

(%)

C

200 nm

OR NP

15

10

5

0103102

Diameter (nm)101 104

20

25

Inte

nsity

(%)

D

–10

–20

–30

0 PR N

P

OR

NP

Zeta

-Pot

entia

l (m

V)

–40

E

120

90

60

150

Cum

ulat

ive

Rele

ase

of R

AP (%

)

30

00 60504030

Time (h)2010 70

PR NP at 0 mM H2O2

OR NP at 1.0 mM H2O2

OR NP at 0 mM H2O2

PR NP at 1.0 mM H2O2

FControl

PCy5 NP2 h1 h 8 h Control

OCy5 NP2 h1 h 8 h

GControl

PCy5 NP2 h1 h 8 h Control

OCy5 NP2 h1 h 8 h

H

100

80604020

0

Cy5

100

106104

PCy5 NP

102

Coun

ts

100

80604020

0

100

106104

OCy5 NP

102

300

200

100

0

2Time (h)

10

***

8

400

MFI

(x10

3 )

*****

PCy5 NP OCy5 NP0 h 1 h 2 h 8 h

I750

500

250

0

2Time (h)

10

***

8

1000

MFI

(x10

3 ) ******

PCy5 NP OCy5 NP0 h 1 h 2 h 8 h

100

80604020

0

Cy5

100

106104

PCy5 NP

102

Coun

ts

100

80604020

0

100

106104

OCy5 NP

102

(A) Schematic of RAP nanotherapies preparation. TEM images (left) and size distribution (right) of PLGA (B) and OxbCD (C) nanotherapies. (D) Zeta-potential values.

(E) In vitro release profiles of PR NP and OR NP. Fluorescence images illustrating internalized Cy5-labeled NPs in VSMCs (F) or macrophages (G). Scale bars indicate 20

mm. Flow cytometric curves (left) and quantification (right) of internalized NPs in VSMCs (H) or macrophages (I). Data are mean � SD (H, I, n ¼ 3). **p < 0.01; ***p <

0.001. Cy5 ¼ Cyanine5; DSPE-PEG ¼ a lipid-polyethylene glycol conjugate; MFI ¼ mean fluorescence intensity; NPs ¼ nanoparticles; OCy5 NP ¼ Cy5-labeled OxbCD

nanoparticle; OR NP ¼ RAP-containing OxbCD nanotherapy; OxbCD ¼ oxidation-responsive b-cyclodextrin material; PCy5 NP ¼ Cy5-labeled PLGA nanoparticle;

PLGA ¼ poly(D,L-lactide-co-glycolide); PR NP ¼ PLGA-derived rapamycin nanotherapy; RAP ¼ rapamycin; TEM ¼ transmission electron microscopy; VSMCs ¼ vascular

smooth muscle cells.



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FIGURE 2 In Vitro Activities of Nanotherapies in VSMCs

AControl Model OxbCD NP RAP PR NP OR NP

1.5

1.0

0.5

2.0 ***

Rela

tive

Calc

ifica

tion

0.0

Cont

rol

PR N

PRA

PO

xbCD

NP

Mod

el

OR

NP

Cont

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PR N

PRA

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xbCD

NP

Mod

el

OR

NP

** ***

B C

90

60

30

120

Cell

Viab

ility

(%)

0

***

DControl Model OxbCD NP RAP PR NP OR NP Tempol

E2520

1015

5

30

MFI

(x10

3 )

Cont

rol

PR N

PRA

PO

xbCD

NP

Mod

el

OR

NPTe

mpo

l0

***

***

G

40

2030

10

50

Apop

totic

Cel

ls (%

)

Cont

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PR N

PRA

PO

xbCD

NP

Mod

el

OR

NPTe

mpo

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H

90

3060

120

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(%)

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PR N

PRA

PO

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NP

Mod

el

OR

NPTe

mpo

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FModel

0.6 11.0

65.2 23.2

OxbCD NP

0.5 6.6

72.8 20.1

RAP

1.0 14.2

75.6 9.2

PR NP

2.3 12.1

78.2 7.4

OR NP

0.2 1.1

97.0 1.7

Tempol

0.1 1.2

96.7 2.0

Control

0.1 1.2

97.7 1.0

Annexin V-FITC

7-AA

D

(A) Visualization of calcium deposition via Alizarin Red staining. Upper panel, digital photos; lower panel, microscopic images of VSMCs. Quantified calcium deposition

(B) and cell viability (C) of VSMCs. (D) Fluorescence images of DHE-stained VSMCs after H2O2 stimulation. Scale bars, 20 mm. (E) Quantified fluorescent intensities of

DHE showing intracellular ROS generation. Flow cytometric profiles (F) and quantitative data (G) of apoptotic VSMCs after induction with H2O2. (H) Cell viability after

different pre-treatments and stimulation with H2O2. Cells in the control group were treated with fresh medium. Data are mean � SD (n ¼ 3). *p < 0.05; **p < 0.01;

***p < 0.001. DHE ¼ dihydroethidium; ROS ¼ reactive oxygen species; other abbreviations as in Figure 1.


2595

the model group (Figures 2A and 2B). Pre-treatmentwith RAP or nanotherapies dramatically reducedcalcification. OR NP–treated cells displayed thelowest calcium deposition. Furthermore, treatmentwith different RAP formulations protected VSMCsfrom Ca/Pi-induced cell death (Figure 2C), with themost efficacious effect for the OR NP group.

NANOTHERAPIES DECREASE H2O2-INDUCED ROS

GENERATION AND APOPTOSIS IN VSMCs. VSMCstreated with H2O2 showed notably increased intra-cellular ROS (Figure 2D). Pre-incubation with RAPformulations significantly reduced ROS. The respon-sive nanotherapy OR NP even completely inhibitedthe production of ROS, comparable to the effect ofTempol, a frequently used ROS scavenger. Thisobservation was affirmed by flow cytometric quanti-fication (Figure 2E, Online Figure 3). Accordingly, ourresults demonstrated that the antioxidative stress

activity of RAP was notably potentiated by loadinginto nanocarriers, particularly for the responsive RAPnanotherapy.

Mechanistically, depletion of medial VSMCs byapoptosis contributes to the growth of AAAs (7). H2O2

could induce significant cell apoptosis (Figures 2F and2G). Pre-incubation with RAP formulations attenu-ated H2O2-induced VSMCs apoptosis. At the samedose of RAP, the responsive nanotherapy exhibitedthe most beneficial effect. Consistently, OR NP mosteffectively maintained VSMCs viability (Figure 2H).Consequently, OR NP can significantly inhibit H2O2-induced VSMCs apoptosis by reducing intracellularROS generation.

IN VIVO TARGETING CAPABILITY OF NANOPARTICLES.

In vivo targeting of NPs to aneurysmal aortas wasexamined in rats with CaCl2-induced AAAs, usingCy7.5-labeled NPs based on PLGA (PCy7.5 NP) or




2596

OxbCD (OCy7.5 NP) (Online Figure 4A). At 8 h after IVinjection, fluorescent signals were clearly observed atthe injury site of abdominal aortas isolated from AAArats (Figure 3A). Quantitative analysis showed signif-icantly higher accumulation of OCy7.5 NP than that ofPCy7.5 NP. By contrast, the abdominal aortas fromnormal rats treated with fluorescent NPs displayed nosignificant fluorescence. Besides, distribution of NPsin liver, spleen, and lung were observed (OnlineFigure 4B). Fluorescence observation on cry-osections confirmed the accumulation of NPs inaneurysmal aortas (Figure 3B), with more significantfluorescence in the OCy5 NP group. These resultsdemonstrated that IV administered NPs are able topassively target aneurysmal aortas.

TREATMENT OF AAAs BY NANOTHERAPIES IN

RATS. We examined in vivo efficacy of RAP nano-therapies in AAA rats. Treatments by IV injectionwere conducted at day 2 after CaCl2 stimulation(Online Figure 5A). Hematoxylin and eosin–stainedsections showed that the maximal diameter ofinfrarenal abdominal aortas in the model group wassignificantly enlarged (Online Figure 5B, Figures 3Cand 3D). Among different formulations, OR NP mosteffectively reduced the aneurysm diameter. AlizarinRed–stained sections revealed substantially lowercalcification after treatment with RAP formulations(Online Figure 5B, Figure 3C), particularly in the ORNP group (Figure 3E). Similarly, Verhoeff-Van Giesonstaining indicated the broken and damaged elasticlamina in the model and blank OxbCD NP groups,whereas elastin was preserved after therapy with RAPand nanotherapies. Again, OR NP prevented elastindegradation to a much better degree than PR NP(Online Figure 5B, Figure 3C).

Immunohistochemistry analyses showed that thelevels of MMP-2 and MMP-9, 2 important MMPsinvolved in AAAs (3), were remarkably decreased byRAP formulations, especially OR NP (Figure 3F, OnlineFigure 5C). Consistently, quantification by Westernblotting revealed their expression in aneurysmalaortas was most significantly decreased after OR NPtreatment (Figures 3G and 3H). Compared with bothmodel and OxbCD NP groups, infiltration of CD68þ

macrophages in the adventitia and ruptured mediawas attenuated by OR NP (Figure 3F, Online Figure 6).CD31 staining indicated the most intact endotheliumfor OR NP–treated rats. These results demonstratedthat the ROS-responsive nanotherapy can substan-tially inhibit the development of AAAs.

To address the mechanisms responsible for thebeneficial effects of nanotherapies, the levels of

representative inflammatory cytokines and chemo-kines in aortas were determined. The expressions oftumor necrosis factor (TNF)-a, interferon (IFN)-g,interleukin (IL)-1b, and monocyte chemoattractantprotein (MCP)-1 were dramatically reduced in bothnanotherapy groups, when compared with the modelgroup (Online Figure 7A). Also, nanotherapy-treatedgroups showed notably lower levels of ROS in aneu-rysmal tissues (Online Figures 7B to 7D).

In a separate study, OR NP was administered at day10 after CaCl2-induced injury. In this case, beneficialefficacies were also achieved, as implicated bysignificantly inhibited aneurysmal expansion andcalcification, remarkably decreased elastin degrada-tion, as well as notably reduced inflammatoryresponse and oxidative stress (Online Figure 8).

Taken together, RAP nanotherapies, especially ORNP can significantly prevent the progression of AAAsin rats, by reducing expressions of MMPs and proin-flammatory cytokines/chemokines, inhibiting calcifi-cation, and lowering ROS generation.

ENGINEERING OF A CRGDFK-FUNCTIONALIZED

RESPONSIVE NANOTHERAPY. To develop a nano-therapy with enhanced targeting, we functionalizedthe responsive nanotherapy with cRGDfK, a cyclo-peptide ligand that can target neovessels via bindingwith avb3 integrin (19). cRGDfK was first covalentlylinked with a lipid-PEG conjugate DSPE-PEG (OnlineFigure 9). The ROS-responsive RAP nanotherapywith cRGDfK decoration (referred to as ROR NP)(Figure 4A), was also prepared by the aforementionednanoprecipitation method. ROR NP showed a core-shell spherical shape (Figures 4B and 4C), with themean diameter of 179 � 3 nm and zeta-potentialof �25 mV. The RAP content in ROR NP was 8.1 wt%.ROR NP also displayed H2O2-triggered drug releasekinetics (Figure 4D). This result is consistent with thehydrolysis profiles of blank NPs (Online Figure 10A).

IN VITRO EVALUATIONS OF THE CRGDFK-DECORATED

NANOTHERAPY. Blank OxbCD NPs with or withoutcRGDfK showed comparable cell viability in VSMCs(Online Figure 10B). Using Cy5-labeled fluorescent NP(ROCy5 NP) (Online Figure 11A), we found time-dependent internalization of cRGDfK-coated NP inmacrophages and VSMCs (Online Figures 11B to 11E,Figures 4E and 4F). Of note, cRGDfK-functionalizedNP exhibited significantly higher cellular uptake ascompared with the undecorated control in both cells.

Although both ROS-responsive nanotherapiessignificantly inhibited Ca/Pi-induced calcification andcell death in VSMCs (Figures 4G to 4I), ROR NPshowed more beneficial effects. As compared with OR




















FIGURE 3 In Vivo Targeting and Efficacies of Nanotherapies in AAA Rats

BSaline PCy5 NP OCy5 NP

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.5 N

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el

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tive

MM

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ents ***

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G

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al

MMP-2

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PR N

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el

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NP

(A) Ex vivo images (left) and quantitative analysis (right) showing the accumulation of PCy7.5 NP or OCy7.5 NP in normal (AAA-) or aneurysmal (AAAþ) aortas at 8 h

after intravenous injection. (B) Fluorescence images illustrating PCy5 NP or OCy5 NP in abdominal aortas. Scale bars indicate 20 mm. (C) Histological sections indicating

the maximal diameter, calcium deposition, and elastin degradation in aneurysmal aortas. The mean maximal diameter (D) and calcium contents (E) of abdominal aortas.

(F) Immunohistochemistry analysis of the levels of MMP-2 and MMP-9 as well as the counts of macrophages and vascular endothelial cells in aneurysmal aortas.

Western blot bands (G) and quantitative analysis (H) of MMP-2 and MMP-9 levels. In the normal group, healthy rats were treated with saline. Data are mean � SD

(n ¼ 3). *p < 0.05; **p < 0.01; ***p < 0.001. Cy7.5 ¼ Cyanine7.5; OCy7.5 NP ¼ Cy7.5-labeled OxbCD nanoparticle; PCy7.5 NP ¼ Cy7.5-labeled PLGA nanoparticle;

VVG ¼ Verhoeff-Van Gieson; other abbreviations as in Figure 1.


2597

FIGURE 4 Engineering of a ROS-Responsive, cRGDfK Targeting Nanotherapy ROR NP

MModel ROR NPOR NP

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(x10

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el

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(%)

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ive

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ROR NP at 0 mM H2O2

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2Time (h)

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OCy5 NPROCy5 NP

F GModel ROR NPOR NP

(A) Schematic of ROR NP. (B) TEM image (left) and size distribution (right) of ROR NP. Zeta-potential (C) and in vitro release profiles (D) of ROR NP. (E) Fluorescence

images of VSMCs incubated with ROCy5 NP or OCy5 NP for different periods of time. (F) Quantified fluorescence intensities by flow cytometry. (G) Ca/Pi-induced

calcification of VSMCs pre-treated with different formulations. Upper panel, digital photos; lower panel, microscopy images of VSMCs. Quantification of the calcium

content (H) and cell viability (I) of VSMCs pre-treated with different formulations and induced with Ca/Pi. Flow cytometric profiles (J) and quantitative analysis (K) of

apoptotic cells as well as cell viability (L) of VSMCs pre-treated with nanotherapies and stimulated with H2O2. (M) Fluorescence images showing intracellular ROS

generation. (N) Fluorescence intensity of DHE quantified by flow cytometry. Scale bars indicate 20 mm. Data are mean � SD (n ¼ 3). *p < 0.05; **p < 0.01; ***p <

0.001. cRGDfK ¼ a cyclic peptide ligand; ROCy5 NP ¼ Cy5-labeled OxbCD nanoparticle with cRGDfK decoration; ROR NP ¼ a ROS-responsive, cRGDfK targeting RAP

nanotherapy; other abbreviations as in Figures 1 and 2.



2598


2599

NP, ROR NP more significantly suppressed apoptosis,maintained cell viability, and decreased ROS genera-tion in VSMCs induced by H2O2 (Figures 4J to 4N,Online Figure 11F). Together, these results demon-strated that ROR NP can further potentiate the ther-apeutic effects of RAP by enhancing intracellulardelivery of drug molecules via receptor-mediatedendocytosis.

IN VIVO TARGETING AND EFFICACIES OF THE

CRGDFK-FUNCTIONALIZED NANOTHERAPY IN

RATS. At 8 h after IV injection of Cy7.5-labeledcRGDfK-coated NP (ROCy7.5 NP) in AAA rats (OnlineFigures 12A and 12B), ex vivo imaging showedevident fluorescent signals at aneurysmal sites(Figure 5A), distinctly different from those of normalrats. The fluorescent intensity of cRGDfK-coated NPwas 2.5-fold higher than that of cRGDfK-deficient NP.Nevertheless, nearly similar distribution profiles werefound in major organs (Online Figure 12C). Furtherimmunofluorescence examination indicated theconsiderable distribution of Cy5-labeled NP in thediseased sites of abdominal aortas (Figure 5B, OnlineFigure 13). Notably, cRGDfK-coated NP displayedrelatively high accumulation in both intimal andmedial regions. These results substantiated that theaneurysmal targeting capacity of responsive NP canbe notably improved by decoration with cRGDfK.

In vivo efficacies were then assessed in AAA rats(Online Figure 14A). ROR NP more significantlyreduced the maximum diameter of abdominal aortas,decreased calcification, and suppressed elastindegradation, when compared with the control nano-therapy OR NP (Online Figure 14B, Figures 5C to 5E). Ascompared with OR NP, ROR NP had more prominenteffects on decreasing the MMP-2 and MMP-9 levels,lowering macrophage infiltration, and preservingendothelial integrity (Figures 5F to 5H, OnlineFigures 14C and 14D). Likewise, ROR NP more signifi-cantly reduced the levels of TNF-a, IFN-g, IL-1b,MCP-1,and ROS in abdominal aortas (Figure 5I, OnlineFigure 15). Of note, the efficacious discrepancy of ORNP between this and aforementioned studies shouldbe due to different batches of model animals. Collec-tively, ROR NP can more significantly prevent thedevelopment of AAAs by actively targeting aneurysms.

A ROS-RESPONSIVE NANOTHERAPY SIMULTANEOUSLY

FUNCTIONALIZED WITH CRGDFK AND MACROPHAGE

CELL MEMBRANE. To further improve targeting capa-bility of the cRGDfK-decorated nanotherapy, it wasfunctionalized by macrophage cell membrane(Figure 6A), in view of the fact that local macrophageinfiltration is one of the main pathological mecha-nisms of AAA (2). As a proof of concept, rat alveolar

macrophage NR8383 cells were used. CMVs(w300 nm) were first prepared from NR8383 cells(Figure 6B). By coextruding CMVs and ROR NP, amacrophage-mimetic, active targeting, and ROS-responsive RAP nanotherapy (CROR NP) was fabri-cated, exhibiting a typical core–shell structure(Figure 6B). CROR NP showed negative zeta-potential,comparable to that of CMVs (Figure 6C). CROR NPdisplayed narrow size distribution (Figure 6D), withan average diameter of 190 nm. The successfulcoating of macrophage cell membrane was confirmedby profiling protein components. In this case, CRORNP showed similar protein bands as CMVs (Figure 6E).The RAP content in CROR NP was 4.1 wt%. In com-parison to that of ROR NP, RAP release from CROR NPwas slightly delayed, resulting from the presence ofouter membrane that acts as a diffusion barrier fordrug molecules (Figure 6F). This is consistent with thehydrolysis behaviors of blank NPs (OnlineFigure 16A). These results demonstrated the suc-cessful development of a cRGDfK and macrophagecell membrane-functionalized ROS-responsivenanotherapy.

IN VIVO TARGETING AND EFFICACIES OF CROR NP

IN RATS. Using cRGDfK/cell membrane functional-ized and Cy7.5-labeled NP (CROCy7.5 NP) (OnlineFigure 16B), in vivo targeting capability was exam-ined. Compared with the control ROCy7.5 NP, stron-ger fluorescent signals were detected for CROCy7.5NP at AAA sites (Figure 6G). Almost no fluorescenceappeared in aortas of normal rats receiving the samedose of CROCy7.5 NP. Also, fluorescent signals wereobserved in liver, spleen, and lung for the ROCy7.5 NPand CROCy7.5 NP groups (Online Figure 16C).Different NPs exhibited similar pharmacokineticprofiles post-IV injection in healthy or AAA rats(Online Figures 16D and 16E). As compared with otherNPs, CROCy7.5 NP displayed prolonged blood circu-lation. The elimination half-life of CROCy7.5 NP inAAA rats was slightly longer than that in healthy rats(Online Figure 16F).

Using Cy5-labeled and cell membrane–coated NP(CROCy5 NP) (Online Figure 16G) and the controlROCy5 NP, we also observed the localization of bothNPs in aneurysms of abdominal aortas (Figure 6H).CROCy5 NP exhibited notably stronger fluorescentsignals, agreeing with ex vivo images. These resultscollectively demonstrated that macrophage cellmembrane cloaking can additionally enhance theactive targeting capacity endowed by cRGDfKfunctionalization.

After IV treatment (Online Figure 17A), CROR NPshowed greater antianeurysmal activity than ROR NP,






















FIGURE 5 In Vivo Targeting and Therapeutic Activities of ROR NP in AAA Rats

Salin

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(A) Ex vivo images (left) and histograms (right) showing accumulated ROCy7.5 NP or OCy7.5 NP in aneurysmal aortas. (B) Immunofluorescence analysis of

localized ROCy5 NP in aneurysms. Scale bars indicate 20 mm. (C) Hematoxylin and eosin, Alizarin Red, or VVG stained histological sections of aneurysmal

aortas. Quantification of the maximal diameter (D) and calcium contents (E) of abdominal aortas. (F) Immunohistochemistry analyses of aneurysmal aortas.

Representative Western blot bands (G) and quantitative analysis (H) of MMP-2 and MMP-9 levels in abdominal aortas. (I) The levels of proinflammatory

cytokines/chemokines in abdominal aortas. Data are mean � SD (n ¼ 3). *p < 0.05; **p < 0.01; ***p < 0.001. ROCy7.5 NP ¼ Cy7.5-labeled OxbCD

nanoparticle decorated with cRGDfK; SMA ¼ smooth muscle actin; other abbreviations as in Figures 1 to 4.



2600

FIGURE 6 Development of CROR NP and Its In Vivo Targeting Capability

EC

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CROR NP at 0.0 mM H2O2



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(A) Schematic of CROR NP. TEM images (B) and zeta-potential values (C) of CMVs and CROR NP. (D) Size distribution of CROR NP. (E) SDS-polyacrylamide gel

electrophoresis analysis of proteins on ROR NP, CMVs, and CROR NP. (F) In vitro release profiles of ROR NP or CROR NP. (G) Ex vivo images (left) and quantification

(right) of accumulated ROCy7.5 NP or CROCy7.5 NP in aneurysmal aortas. (H) Immunofluorescence observation of localized ROCy5 NP or CROCy5 NP. Scale bars indicate

20 mm. Data are mean � SD (n ¼ 3). *p < 0.05; ***p < 0.001. CMV ¼ cell membrane vesicle; CROR NP ¼ a ROS-responsive, cRGDfK targeted, and macrophage cell

membrane-coated rapamycin nanotherapy; CROCy5 NP ¼ Cy5-labeled OxbCD nanoparticle with cRGDfK and cell membrane coating; CROCy7.5 NP ¼ Cy7.5-labeled

OxbCD nanoparticle with cRGDfK and cell membrane coating; other abbreviations as in Figures 1 to 5.


2601

in terms of attenuating the expansion of the aorticdiameter, preventing calcification, decreasing elastindegradation, and maintaining endothelial integrity,as well as dampening inflammation and loweringoxidative stress in aneurysmal tissues (Figure 7,Online Figures 17 and 18). The potentiated therapeu-tic effects of CROR NP were correlated with improvedtargeting after macrophage membrane cloaking.

IN VIVO SAFETY STUDIES. Safety tests were per-formed in rats (Online Figures 19 and 20). During14 days of inspection, normal behaviors were

observed for all rats. All examined animals displayedcomparable body weight gain and the organ index.We did not find remarkable changes in representativehematological parameters in nanotherapy-treatedgroups. No abnormal variations in typical bio-markers associated with hepatic and kidney functionswere detected for different groups. Likewise, we didnot observe injuries or infiltration of inflammatorycells in hematoxylin and eosin–stained sections ofmajor organs. These preliminary data indicated thatdifferent RAP nanotherapies are safe for IVadministration.



FIGURE 7 In Vivo Efficacy of CROR NP in AAA Rats

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tent

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B

Diam

eter

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) * ***3

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2

0

Mod

el

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NP

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(A) Hematoxylin and eosin, Alizarin Red, and VVG stained histopathological sections of aneurysmal aortas. The quantified maximal diameter (B) and calcium contents

(C) of abdominal aortas. (D) Immunohistochemistry analyses of MMP-2 and MMP-9 levels, the macrophage content, and the vascular endothelial integrity. Western

blot bands (E) and quantitative data (F) of MMP-2 and MMP-9. (G) The levels of TNF-a, IFN-g, 1L-1b, and MCP-1 in abdominal aortas. Data are mean � SD (n ¼ 3). *p <

0.05; **p < 0.01; ***p < 0.001. Abbreviations as in Figures 1 to 6.



2602

DISCUSSION

Despite worldwide efforts over the past decades,there remain no effective drugs that can prevent thegrowth of aneurysms and delay aneurysm rupture inthe clinical setting (8,15,20). To develop an effectivenanotherapy for aneurysms, we established a hierar-chical functionalization approach, using RAP as acandidate drug, for which clinical applications havebeen limited due to its adverse effects, such asimmunosuppression and increased lipid levels (21).Therefore, targeted delivery strategies that canreduce side effects of RAP while maintaining or even

amplifying its efficacy are desperately required(Central Illustration).

According to abnormally increased ROS in aneu-rysms (4), we first constructed a ROS-responsivenanoplatform using an oxidation-labile materialOxbCD to achieve triggerable drug delivery. TheOxbCD-derived nanotherapy OR NP can release RAPupon triggering by high levels of ROS. By internali-zation and responsively releasing RAP, OR NP effec-tively attenuated Ca/Pi-induced calcification inVSMCs and significantly inhibited intracellular ROSgeneration and cell apoptosis induced by H2O2. NPsbased on OxbCD were considerably accumulated in

CENTRAL ILLUSTRATION Targeted Treatment of AAA by a Multifunctional Nanotherapy

Cheng, J. et al. J Am Coll Cardiol. 2018;72(21):2591–605.

A hierarchically engineered, ROS-responsive, integrin targeting, and macrophage-mimetic rapamycin nanotherapy can efficaciously prevent the development of AAAs

by effectively targeting aneurysms after intravenous delivery. CROR NP ¼ a ROS-responsive, cRGDfK targeted, and macrophage cell membrane-coated rapamycin

nanotherapy; ROS ¼ reactive oxygen species; VSMC ¼ vascular smooth muscle cell.


2603

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE:

Overproduction of ROS in aneurysmal tissue might

serve as a molecular basis for targeted treatment. In

preliminary studies in vitro and in vivo, a ROS-

responsive nanoparticle, in combination with a

peptide ligand for integrin and macrophage cell

membranes, released rapamycin molecules in

response to the inflammatory microenvironment,

slowing abdominal aortic aneurysm progression.

TRANSLATIONAL OUTLOOK: Additional preclini-

cal studies are needed to confirm the therapeutic

benefit of targeted nanotherapy with rapamycin or

other antiproliferative agents and assess potential

toxicity during long-term treatment as a prelude to

clinical trials.



2604

aneurysms of AAA rats. This was achieved by themicrodefects in the destructed aneurysmal wall andthe damaged endothelial layer (14,22). In addition,neovessels in the media and adventitia mightcontribute to the accumulation of NPs in aneurysms(23). Moreover, ROS-triggered oxidation of OxbCDgenerates phenylboronic acid that can adhere to cellsor extracellular matrix at inflamed sites (24). Theseeffects collectively accounted for the high targetingefficiency of OxbCD NP. Consistently, the responsivenanotherapy displayed more desirable efficacy thanthat of the PLGA nanotherapy.

To further enhance targeting efficiency of theresponsive nanotherapy, we functionalized OR NP bysurface decoration with a cyclic peptide ligandcRGDfK. The cRGDfK-functionalized nanotherapyROR NP showed significantly potentiated in vitro ac-tivities in VSMCs. Also, cRGDfK coating increased theaccumulation of NPs in aneurysms in rats, therebyleading to additionally enhanced in vivo efficacies.Our results for the first time demonstrated thatcRGDfK can be used for targeted therapy of AAAs.

Finally, a macrophage-mimetic approach was usedto functionalize the cRGDfK-coated nanotherapy.Coating via cell membranes is an effective strategy toenhance delivery efficiency of various nanovehicles(25,26). Considering the critical role of macrophagesin the pathogenesis of AAAs (2), macrophage mem-brane was used to cloak cRGDfK-functionalizedresponsive NPs. We found that surface coating withmacrophage membrane further promoted aneurysmtargeting of cRGDfK-armed OxbCD NP, resulting fromhomotypic and heterotypic cell adhesion mechanisms(27). The macrophage-like recruitment effect mightalso contribute to the increased delivery efficiency atAAA sites (2). Correspondingly, the macrophage-mimetic, cRGDfK targeting nanotherapy CROR NPinhibited the progression of AAAs to a significantlygreater extent than ROR NP without membranecoating. Importantly, preliminary in vivo tests sub-stantiated that our nanotherapies displayed goodsafety profile after IV administration. In practice,based on screening and ultrasound surveillance ofaneurysm size, ROS might be used as biomarkers todetermine the right time point for treatment withnanotherapies. Also, these nanotherapies can becombined with other traditional anti-aneurysmaldrugs to prevent aneurysm formation. To addressmacrophage cell membrane limitations for trans-lation, the plasma membrane–derived proteins

responsible for aneurysm targeting will be incorpo-rated into the lipid shell of ROR NP in follow-upstudies (28).

STUDY LIMITATIONS. The therapeutic benefits andsafety profiles of our targeting nanotherapies need tobe examined in different animal models of AAAs, withlong-term treatment. Furthermore, whether this tar-geting strategy can be generalized to other cardio-vascular drugs remains to be addressed.

CONCLUSIONS

We developed a ROS-triggerable, aneurysmal target-ing nanotherapy via a hierarchical functionalizationstrategy, according to the pathophysiology of aneu-rysms. By effective delivery of the loaded therapeuticmolecules to diseased aortic sites, this nanotherapyefficaciously prevented aneurysm expansion in AAArats.

ADDRESS FOR CORRESPONDENCE: Prof. HouyuanHu, Department of Cardiology, Southwest Hospital,Third Military Medical University, 30 Gaotanyan MainStreet, Chongqing 400038, China. E-mail:[email protected]. OR Prof. Jianxiang Zhang,Department of Pharmaceutics, College of Pharmacy,Third Military Medical University, 30 Gaotanyan MainStreet, Chongqing 400038, China. E-mail: [email protected].

mailto:[email protected]




2605

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KEY WORDS aneurysm, inflammation,nanotherapy, reactive oxygen species,targeting

APPENDIX For an expanded Methodssection as well as supplemental figures, pleasesee the online version of this paper.

http://refhub.elsevier.com/S0735-1097(18)38631-5/sref1


















































































































a targeting nanotherapy for abdominal aortic aneurysms · 2018-11-14 · a targeting nanotherapy...

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