Biofunctional PolymerâLipid Hybrid High-Density Lipoprotein

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Biofunctional polymer-lipid hybrid high density lipoproteinmimicking nanoparticles loading anti-miR155 for combined antiatherogenic effects on macrophages Jing Lu, Yi Zhao, Xiaoju Zhou, Jian Hua He, Yun Yang, Cuiping Jiang, Zitong Qi, Wenli Zhang, and Jianping Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00436 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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4

Biofunctional polymer-lipid hybrid high density lipoprotein-mimicking nanoparticles loading anti-miR155 for combined antiatherogenic effects on macrophages

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Jing Lu, Yi Zhao, Xiaoju Zhou, Jian Hua He, Yun Yang, Cuiping Jiang, Zitong Qi,

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Wenli Zhang and Jianping Liu*

7

Department of Pharmaceutics, China Pharmaceutical University, Nanjing, PR China

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KEYWORDS

9

Biofunction, polymer-lipid hybrid HNP, acid-labile PEI, anti-miR155, combination

1 2 3

10

therapy, atherosclerosis.

11

ABSTRACT

12

A biofunctional polymer-lipid hybrid high density lipoprotein-mimicking

13

nanoparticle

(HNP)

loading

anti-miR155

was

constructed

14

antiatherogenic effects on macrophages. The HNP consisted of an anti-miR155 core

15

condensed by acid-labile polyethylenimine (acid-labile PEI) polymers and a lipid

16

bilayer coat that was decorated with apolipoprotein A-1, termed acid-labile PEI/HNP.

17

The acid-labile PEI was synthesized with low molecular weight PEI and

18

glutaraldehyde to reduce the cytotoxicity and facilitate nucleic acids escaping from

19

acidic endolysosomes. The increased silencing efficiency of acid-labile PEI/HNP was

20

ascribed to the clathrin-mediated endocytosis and successful endolysosomal escape.

21

Decreased intracellular reactive oxygen species production and DiI-oxLDL uptake

1

ACS Paragon Plus Environment

for

combined

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revealed the antioxidant activities of both anti-miR155 and HNP. Cholesterol efflux

23

assay indicated the potential of HNP in reverse cholesterol transport. Collectively, the

24

acid-labile PEI/HNP not only realized the efficacy of anti-miR155 in macrophages,

25

but also exerted the anti-atherosclerotic biofunction of HNP.

26

INTRODUCTION

27

Atherosclerosis is a chronic inflammatory lesion with high morbidity and

28

mortality worldwide, which is caused by excess lipids deposition in the arterial blood

29

vessels.1 Macrophages situated within the intima play a pivotal role in atherogenic

30

programming, which internalize modified lipoproteins resulting in foam cells

31

formation and further promote the retention of lipoproteins. Excess lipids

32

accumulation and defective phagocytic clearance contribute to the formation of a

33

necrotic core, triggering myocardial infarction or stroke.2,3 The macrophage is

34

therefore pursued as a therapeutic target to prevent atherosclerosis events.

35

MicroRNA (miRNA)-based therapy has been increasingly recognized as a

36

promising therapeutic approach for atherosclerosis.4,5 MicroRNA-155 (miR155), a

37

class of endogenous noncoding RNA with about 23 nucleotides, has been reported to

38

be expressed specifically in macrophages of atherosclerotic plaques.6 As a multi-target

39

molecule, miR155 induces atherosclerosis by base paring with different target

40

mRNAs, causing series of impaired inflammatory responses and lipid metabolisms.

41

Tian et al. proved that miR155 promoted lipid uptake and reactive oxygen species

42

(ROS) production of macrophages to induce foam cells formation, and miR155

43

deficiency in apolipoprotein E-deficient (apo E−/−) mice mitigated atherogenesis via

2


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reducing inflammatory responses of macrophages and enhancing macrophage

45

cholesterol efflux.6-8 Therefore, anti-miR155 with complementary sequences against

46

miR155 function in macrophages may be a promising strategy to halt atherogenesis.

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However, free anti-miRNA is unstable in the plasma and may suffer nucleases

48

degradation and renal clearance.9 It is urgent to find an effective carrier for the direct

49

anti-miR155 delivery into macrophages.

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Endogenous high density lipoproteins (HDLs) are dynamic natural nanoparticles,

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existing mainly in two distinct structures, discoidal HDL and spherical HDL.10 The

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discoidal HDL is comprised of a phospholipid bilayer circumscribed with two

53

apolipoprotein A-1 (apo A1) molecules, and the spherical HDL has a hydrophobic

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lipids core surrounded by a phospholipid monolayer with apo A1 embedded. Apo A1,

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the most important protein constituent of HDL, is proved to be with high affinity to

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scavenger receptor type B-1 (SR-B1) expressed abundantly in macrophages and

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cancer cells.10 Based on the long circulation half-life, excellent biocompatibility and

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SR-B1-specific targeting, considerable interests have been focused on utilizing

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recombinant HDL nanoparticles (rHDL) for the delivery of nucleic acids. Biomimetic

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rHDL nanocarriers with cholesterylated nucleic acids adsorbed onto the surface for

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the direct cytosolic delivery of cargos achieved improved gene silencing.11-14

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McMahon constructed HDL inspired-hybrid vehicles through assembling templated

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lipoprotein particles with single-stranded RNA complements of short-interfering RNA

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(siRNA) after formulation with a cationic lipid whereby RNA was also localized to

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the particle surface.15 Shahzad et al. incorporated siRNA into rHDL, facilitating

3


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highly efficient systemic delivery mediated by the over-expressed SR-B1 in cancer

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cells.16 These contents make the rHDL nanocarriers appealing for anti-miR155

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delivery.

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In addition to the superiority as nanocarriers, rHDL has been explored as a

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potential anti-atherosclerosis therapeutics.17,18 Intravenous infusion of rHDL has been

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shown to have an anti-atherosclerotic effect in animals19-21 and human models22,23.

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The benefits of rHDL therapy appear to result from the reverse cholesterol transport

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(RCT) pathway in much the same way as endogenous HDL.24,25 HDL removes

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cholesterol from the lipid-loaded foam cells via efflux through their surface receptors,

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ATP-binding cassette subfamily members A1 (ABCA1), G1(ABCG1) and SR-B1, and

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then the cholesterol-loaded HDL is transported to the liver for elimination.10

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Moreover, HDL may have benefits beyond RCT in its atheroprotective role, including

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the antioxidant functions26,27, anti-inflammatory effects28,29, and endothelial cells

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protective actions30,31, which have also been manifested in the progress of rHDL

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regressing atherosclerotic plaques.19 We attempted to combine the unique

81

anti-atherosclerotic biological function of rHDL with the anti-miR155 efficacy for

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effective atheroprotection. Notably, rHDL has been tested as a biofunctional

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therapeutic agent for other diseases. For instance, Yang32 prepared gold core HDL

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nanoparticles as therapeutic agents for lymphoma by binding to SR-B1 and interfering

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with cellular cholesterol flux and Numata33 employed nanodiscs as therapeutic

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delivery agents to inhibit respiratory syncytial virus infection in the lung. The intrinsic

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therapeutic effect of HDL nanostructures was defined as “theralivery” by Mutharasan

4


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recently.34

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In this study, we constructed a HDL‐mimicking nanoparticle (HNP) containing a

90

condensed anti-miR155 core and a lipid bilayer envelope structure modified with apo

91

A1 (Scheme 1), providing greater protection for nucleic acids from the degradation

92

and loss in systemic circulation. Due to lacking of the ability of lipids core to

93

condense nucleic acids, a templated anti-miR155 nanoparticle was used as an

94

alternative core inside the HNP. Furthermore, the alternative core would not interfere

95

with the biological functions of HNP which has been demonstrated that HDL

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biomimics with a PLGA core or a gold nanoparticle core were still capable of

97

mediating cholesterol efflux.35,36 The stable core-shell structure allowed a sufficient

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lipidic bilayer space for cholesterol storage.35 Branched polyethylenimine (PEI, 25

99

kDa), a cationic polymer, is often employed to condense nucleic acids with high

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transfection efficiency based on the ‘proton sponge’ hypothesis.37 However, the high

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cytotoxicity of PEI limits the application owing to the interaction of free PEI with

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cellular components which disturbs the normal cellular process after the

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internalization and release of nucleic acids.38 To reduce the cytotoxicity after entering

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the cell, a high molecular weight PEI with acid-labile linkers was synthesized

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between low molecular weight PEI (1.8 kDa) and glutaraldehyde. Then the acid-labile

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PEI was bound to anti-miR155 and inserted into the HNP, termed acid-labile PEI/HNP.

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The polymer of acid-labile PEI was characterized by the

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fourier-transform infrared spectrometer (FT-IR), gel permeation chromatography

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(GPC) and the buffering capacity. The physicochemical properties of acid-labile

5


13

C NMR spectroscopy,

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PEI/HNP, such as the content determination, particle size, zeta potential, morphology

111

and stability test were evaluated. Furthermore, in vitro cell viability, cellular uptake,

112

internalization mechanisms, in vitro gene silencing were assessed in detail. The

113

anti-atherosclerotic efficacies involving the ROS production, DiI-oxidized low

114

density lipoproteins (DiI-oxLDL) uptake, and cholesterol efflux were also validated.

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Scheme 1. Schematics of the acid-labile PEI/HNP.

116 117

2. MATERIALS AND EXPERIMENTAL SECTION

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2.1

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) and block lipid

120

transport 1 (BLT-1) were purchased from Sigma-Aldrich (USA). Branched PEI (1.8

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kDa), heparin sodium, chlorpromazine hydrochloride, genistein, and amiloride were

122

purchased from Aladdin (Shanghai, China). Glutaraldehyde was bought from

123

Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Egg phospholipid (Lipoid

124

S-100) was purchased from Lipoid GmbH (Germany). Quant-iT™ RiboGreen® RNA

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Reagent was purchased from Thermo Fisher Scientific (USA). Cy5.5 mono

Materials.

Branched

PEI

(25

kDa),

cholesterol,

6


sodium

cholate,

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N-hydroxysuccinamide ester (Cy5.5 NHS ester) was from APExBIO (Houston, TX,

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USA). DiI-oxLDL was obtained from Yiyuan Biotech. Co., Ltd (Guangzhou, China).

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RNase A, GelRed, Triton X-100, Hoechst 33342 and ROS Assay Kit were bought

129

from Keygen Biotech Co., Ltd (Jiangsu, China). Lyso-Tracker Red and Radio

130

immunoprecipitation assay (RIPA lysis buffer) were purchased from Beyotime

131

Institute of Biotechnology (Shanghai, China). Raw 264.7 cells were kindly gifted

132

from Atherosclerosis Research Centre, Nanjing Medical University (Nanjing, China).

133

The apo A1 (97% purity) was isolated from the albumin waste as described

134

previously.35 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholest-

135

erol(NBD-cholesterol) was obtained from Cayman Chemical (Michigan, USA).

136

Anti-miR155

(sequence:

137

anti-miRNA

negative

138

5’-ACAACUUAUUACGCACCUA ACUA-3’) and FAM-labled anti-miR155 were

139

synthesized by Genepharm Co., Ltd (Shanghai, China).

140

2.2. Synthesis and Characterization of acid-labile PEI

5’-ACCCCUAUCAC control

(anti-miRNA

AAUUAGCAUUAA-3’), N.C.,

sequence:

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First, 0.6 g PEI (1.8 kDa) dissolved in dichloromethane (0.05 M) was added to

142

the round bottom flask. Then glutaraldehyde (25% aqueous solution, 364 µL)

143

dissolved in dichloromethane (0.005 M) was added dropwise into the clear PEI

144

solution over 2 h under vigorous stirring at room temperature through dropping funnel.

145

After another 20 h stirring, the solvent was removed by evaporation. Subsequently,

146

the collected viscous residue was dissolved in deionized water again and further

147

purified by dialysis in deionized water (MWCO 8000) for 24 h. The dialysate was

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lyophilized for 2 days to achieve yellowish products which was stored at -20 °C until

149

further use.

150

The characteristics of the acid-labile PEI were analyzed by

13

C NMR spectrum

151

(AVANCE AV-300, Bruker, Germany) and FT-IR (Tensor 27, Bruker, Germany). GPC

152

(Shimadzu, Japan) was used to determine the average molecular weight of acid-labile

153

PEI. The mobile phase was ddH2O at the flow rate of 1 mL/min and the column

154

temperature was maintained at 40 °C. Samples of PEG with average molecular

155

weights ranging from 3070-21600 Da were used as standards for calibration.

156

2.3. Degradation and Buffering Capacity of acid-labile PEI

157

GPC was employed to measure the degradation rates of acid-labile PEI under

158

different pH conditions. Five mg/mL acid-labile PEI solution dissolved in deionized

159

water was titrated with HCl to reach the initial pH of 4.5, 5.4 and 7.4, respectively.

160

The average molecular weight of polymer was determined at various time points at

161

37 °C. The half-life of the polymer degradation was defined as the time required for

162

the 50% average molecular weight.

163

The buffering capability of acid-labile PEI was measured by acid-base titration.

164

Briefly, 1 mg/mL polymer dispersed in 0.1 M NaCl was prepared and the initial pH of

165

the polymer solution was adjusted to 10.0 with 1 M NaOH. Then 0.1 M HCl was

166

added dropwise to titrate the solution. The pH values were recorded with a pH Meter

167

(Mettler-Toledo, Switzerland). NaCl solution was titrated in the same manner as a

168

blank control.

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2.4. Synthesis of Cy5.5 Labeled apoA 1

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To label apo A1, 25 mg apo A1 was dissolved in 9 mL of sodium bicarbonate

171

solution (0.1 M, pH 8.3-8.5). Five mg Cy5.5 NHS ester in 1 mL of DMSO was added

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to the apo A1 solution, and then the reaction was stirred for 12 h in the dark at room

173

temperature. The resulting product was dialyzed (MWCO 8000-14000 Da) against

174

PBS (pH 7.4) and lyophilized for further use.

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2.5. Preparation of acid-labile PEI/HNP

176

2.5.1. Preparation of acid-labile PEI core

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Polymer was dissolved in RNase-free water with the final concentration of 1

178

µg/µL. To form the acid-labile PEI core, anti-miR155 solution in RNase-free water

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(0.1 nmol/µL) was added to the polycationic solution slowly at a desired N/P ratio.

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After vortexing for 30 s, the condensed anti-miR155 suspension was incubated at

181

room temperature for 30 min.

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2.5.2. Preparation of acid-labile PEI/HNP

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Lipid film hydration-extrusion method was used to prepare the hybrid

184

nanoparticle by enveloping the acid-labile PEI core into liposome, called acid-labile

185

PEI/lipo. Briefly, the lipid compositions composed of phospholipid and cholesterol

186

(10:1, w/w) dissolved in 1 mL of chloroform were mixed into an eggplant flask,

187

evaporating to dryness under vacuum at 40 °C. Then 1 mL condensed anti-miR155

188

complexation was added to the lipid film for electrostatic binding (lipids:cationic

189

complexation = 6:1, w/w), followed by agitation for 5 min in an ice-bath

190

ultrahomogenizer JY92II (Ningbo, China) to hydrate the lipids. Finally, the acid-labile

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PEI/lipo was obtained by extrusion (Lipex, ATS, Canada) through a 200 nm

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polycarbonate membrane 3 times to further package the polycationic core into the

193

lipid bilayers. Sodium cholate dialysis method was carried out to construct the HNP39.

194

250 µL sodium cholate (7 mg/mL in 20 mM Tris-Hcl 8.0 ) and 500 µL apo A1 (14

195

mg/mL in 20 mM Tris-Hcl 8.0) were added to the acid-labile PEI/lipo successively

196

and the final volume was adjusted to 2 mL. The final mixture was then incubated for

197

12 h by stirring gently at 4 °C, followed by dialysis against pH 8.0 Tris-HCl for 48 h

198

to remove excess sodium cholate.

199

2.6. Quantification of anti-miR155 and apo A1

200

The final acid-labile/HNPs were spun down at 15000 rpm for 30 min (4 °C) and

201

then the concentrated nanoparticles were re-dispersed in the Tris-HCl solution (20

202

mM, pH 8.0). Anti-miR155 was quantified by releasing the free anti-miR155 from

203

acid-labile/HNP via Triton X-100 and heparin. Then the releasing anti-miR155 was

204

stained with Ribogreen dye and measured by a microplate reader (POLARstar Omega,

205

BMG Labtech, Germany) at an excitation and emission wavelength of 480 nm and

206

520 nm. For quantification of apo A1, the acid-labile/HNP was prepared with

207

Cy5.5-apo A1 and analyzed at an excitation and emission wavelength of 675 nm and

208

693 nm. Anti-miR155 and apo A1 were quantified against a standard curve, which

209

was established from each of the fluorescently labeled molecules.

210

2.7. Particle Size, Zeta Potential and Morphology Measurements

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The average particle size and zeta potential of acid-labile PEI core, acid-labile

212

PEI/lipo and acid-labile PEI/HNP were determined by ZetaPlus particle size and zeta

213

potential analyzer (Brookhaven Instruments, USA). Particles were dispersed in

10


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deionized water and measured in triplicate.

215

The morphological observation was performed by transmission electron

216

microscopy (TEM) (H-7650, Hitachi, Japan). Briefly, a drop of the prepared sample

217

was negatively stained with 2% w/v phosphotungstic acid aqueous solution for 10 min

218

on a perforated carbon film-coated copper grid for analysis.

219

2.8. Serum Stability and RNase Protection Assay

220

A serum stability test was performed to investigate the ability of HNP to protect

221

anti-miRNA from serum nuclease degradation. Briefly, acid-labile PEI/HNP and

222

naked anti-miR155 at an anti-miR155 concentration of 0.1 µg/µL were exposed to

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10% and 50% fetal bovine serum (FBS) and then incubated at 37 °C for various time

224

periods.

225

RNase protection assay was performed to evaluate the integrity of anti-miR155

226

released from HNP. RNase A (50 µg/mL, 2 µL) was incubated with samples (0.1

227

µg/µL anti-miR155, 10 µL) at 37 °C for 30 min, 1 h and 2 h. At predetermined time

228

point, RNase A was inactivated with EDTA (3 µL, 25 mM) for 20 min at 37 °C.

229

For both serum stability and RNase protection test, free anti-miR-155 needed to

230

be liberated from nanoparticles. 10% Triton X-100 (v/v) causing destruction of lipid

231

bilayer and 0.1% heparin (w/v) dissociating free anti-miR155 were added to aboved

232

samples and incubated at room temperature for 10 min. Afterwards, aliquots of each

233

sample adding with loading buffer were loaded onto a 1.0% (w/v) agarose gel

234

containing GelRed and then electrophoresed for 15 min at a voltage of 90 V in 1 ×

235

TBE buffer solution.

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2.9. Cell culture and Cell Viability Assay

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Mouse macrophage cell line Raw 264.7 cells were cultured in DMEM

238

supplemented with 10% FBS, 100 U/mL penicillin G sodium and 100 µg/mL

239

streptomycin sulfate at 37 °C and 5% CO2.

240

The cytotoxicity of various complexes was evaluated by MTT assay. Raw 264.7

241

cells were seeded in 96-well plates at a density of 1 × 104 cells per well and incubated

242

overnight. After the removal of media, cells were treated with different complexes for

243

24 h. Twenty µL MTT agent (5 mg/mL in PBS) was then added to each well and

244

incubated for another 4 h. Subsequently, the supernatant was replaced by 150 µL

245

dimethyl sulfoxide (DMSO). The optical density values in each well were measured

246

by a microplate reader (Biotek ELx800, USA) at the absorbance of 570 nm. Untreated

247

cells were used as control and cell viability was defined as a percentage of the

248

untreated cells.

249

2.10. Cellular Uptake Study and Active Targeting of acid-labile PEI/HNP to

250

SR-B1

251

To

assess

the

cellular

uptake

of

different

complexes,

fluorescent

252

FAM-anti-miR155 was delivered into Raw 264.7 cells. Raw 264.7 cells (2 × 105

253

cells/well) were plated in 12-well plates and incubated until the 80% confluence.

254

Then the media were renewed by fresh serum-free DMEM containing naked

255

FAM-anti-miR155 alone, 1.8 kDa PEI core, 25 kDa PEI core, acid-labile PEI core,

256

acid-labile PEI/lipo and acid-labile PEI/HNP at a FAM-anti-miR155 concentration of

257

200 nM and the incubation lasted for 6 h. In order to study the active targeting of

12


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acid-labile PEI/HNP, the SR-B1 receptor was blocked with BLT-1 (dissolved in

259

DMSO), pre-incubated with Raw 264.7 cells at 100 µM for 2 h at 37°C prior to

260

acid-labile PEI core, acid-labile PEI/lipo and acid-labile PEI/HNP treatment. After the

261

treatment for 6 h, the solution was withdrawn and the cells were washed, collected

262

and resuspended with PBS 3 times. The cell fluorescence was analyzed by flow

263

cytometry (MACSQuant, Miltenyi Biotec, Gladbach, Germany) at 493/518 nm and a

264

total of 10,000 events were measured for each sample.

265

2.11. Cellular Trafficking Pathway

266

Raw 264.7 cells (2 × 105 cells/well) were seeded in 12-well plates and incubated

267

for 24 h. Energy-dependent cellular uptake experiments were performed by

268

pre-incubating the cells at 4 °C or with 1 mg/mL NaN3 at 37 °C for 30 min. After this

269

pre-incubation, acid-labile PEI/HNP at a FAM-anti-miR155 concentration of 200 nM

270

was added and incubated for another 6 h at the previous temperature. To investigate

271

the influence of uptake inhibitors on cellular uptake pathway, the cells were

272

pre-incubated for 30 min at 37 °C with different endocytosis inhibitors at the

273

following concentrations: chlorpromazine hydrochloride 10 µg/mL, genistein 54.04

274

µg/mL and amiloride 13.3 µg/mL. Acid-labile PEI/HNP (200 nM) was then added and

275

incubated for 6 h. Subsequently, the cells were washed, harvested, and resuspended 3

276

times with PBS. The mean fluorescence intensity was measured using a flow

277

cytometry. Cells with just the presence of acid-labile PEI/HNP were used as controls.

278

2.12. Intracellular Distribution

279

Raw 264.7 cells (2 × 105) were plated on 15-mm glass-bottom dishes growing

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overnight and then exposed to 200 nM acid-labile PEI/HNP. Lyso-Tracker Red (1.5

281

µM) was added and incubated for 0.5 h at a predetermined time, followed by a wash

282

step with PBS for three times. Then cells were fixed with 4% formaldehyde for 15

283

min and stained with Hoechst 33342 (10 µg/mL) for 10 min. Finally, the cells were

284

washed three times with PBS and imaged using an laser scanning confocal

285

microscope (LSM700, Carl Zeiss, Germany).

286

2.13. Assessment of Gene Silencing

287

For quantitative real-time PCR (qRT-PCR) analysis, Raw 264.7 cells (5×105

288

cells/well) were seeded in 6-well plates for 24 h before experiments. The cells were

289

transfected with naked anti-miR155 alone, 1.8 kDa PEI core, 1.8 kDa PEI/lipo, 1.8

290

kDa PEI/HNP, 25 kDa PEI core, 25 kDa PEI/lipo, 25 kDa PEI/HNP, acid-labile PEI

291

core, acid-labile PEI/lipo and acid-labile PEI/HNP (anti-miR155 concentration of 200

292

nM), respectively. After 6 h of cell incubation, the media were exchanged for fresh

293

media, and the cells were continued to incubate for 48 h.

294

The total mRNA was isolated using TriZol reagent (Invitrogen, Grand Island, NY)

295

according to the manufacturer's protocol. Then the miR155 cDNA was synthesized by

296

MicroRNA reverse transcription Kit (GenePharm, Shanghai, China), and the

297

qRT-PCR reactions (Applied Biosystems, USA) were performed using SYBR Master

298

Mix (GenePharm, Shanghai, China). The relative expression of miR155 was

299

calculated using ∆∆Ct method and normalized with U6 snRNA.

300

2.14. DiI-oxLDL Uptake

301

Raw 264.7 cells were seeded in 12-well plates and transfected with naked

14


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anti-miR155 alone, acid-labile PEI core, acid-labile PEI/lipo and acid-labile PEI/HNP

303

for 48 h. The negative controls were set as acid-labile PEI core N.C, acid-labile

304

PEI/lipo N.C, acid-labile PEI/HNP N.C.. Then the cells were loaded with 40 µg/mL

305

DiI-oxLDL for another 6 h. Blank cells treated with DiI-oxLDL were set as positive

306

controls. At the end of incubation period, the cells were washed, fixed with 4%

307

formaldehyde for 15 min and stained with Hoechst 33342 (10 µg/mL) for 10 min. The

308

images were captured by fluorescence microscope (IX 71, Olympus, Japan).

309

2.15. Intracellular ROS Measurement

310

Intracellular ROS levels were monitored by 2', 7'-Dichlorodihy-drofluorescin

311

diacetate assay (DCFH-DA) probe. Raw 264.7 cells were transfected with naked

312

anti-miR155 alone, acid-labile PEI core, acid-labile PEI/lipo, acid-labile PEI/HNP,

313

acid-labile PEI core N.C., acid-labile PEI/lipo N.C. and acid-labile PEI/HNP N.C.

314

(200 nM) for 48 h and then stimulated with 40 µg/mL oxLDL for 6 h. The cells were

315

subsequently incubated with 10 µM DCFH-DA for 20 minutes at 37 °C, followed by

316

washing with PBS three times. Blank cells treated only with oxLDL or DCFH-DA

317

were used as positive controls and negative controls respectively. The mean

318

fluorescence intensity was analyzed using a flow cytometry operated at an excitation

319

wavelength of 488 nm and an emission wavelength of 525 nm.

320

2.16. Cholesterol Efflux Assay

321

Raw 264.7 cells plated in 24-well plates were stimulated with 40 µg/mL oxLDL

322

and labled with 2 µg/mL NBD-cholesterol for 24 h. The cells were washed and

323

incubated with 200 nM nanoparticles in red-free medium for an additional 48 h. Then

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324

supernatant was collected and the cells were lysed in RIPA lysis buffer. The amounts

325

of cholesterol in media and cells were assayed by a microplate reader (POLARstar

326

Omega, BMG Labtech, Germany) at an excitation and emission wavelength of 485

327

nm and 535 nm. The percentage of cholesterol efflux was calculated as (cholesterol in

328

medium)/(cholesterol in medium + cholesterol in cells) × 100 %.

329

2.17. Statistical Analysis

330

Results were reported as mean ± standard deviation (SD), and a minimum of

331

triplicates were performed in each experiment. Statistical analysis was tested by

332

Student's t-test for two groups and ANOVA for multiple groups. Statistically

333

significant difference was considered at p < 0.05, highly significance was p < 0.01.

334

All statistical analyses were performed by SPSS 19.0 (IBM Corporation, USA).

335

3. RESULTS AND DISCUSSION

336

3.1. Synthesis and Characteristics of acid-labile PEI

337

To reduce the cytotoxicity, degradable PEIs with acid-labile imine linkers were

338

synthesized by Schiff's base reaction between low molecular weight PEI (1.8 kDa)

339

and glutaraldehyde (Scheme 2).38,40 Formation of the polymers was confirmed by 13C

340

NMR spectrum, FT-IR and GPC. As shown in Figure 1A, the appearance of a carbon

341

signal at 164.0582 ppm corroborated the formation of imine linkers in

342

spectrum and the result was also confirmed by the absorbance at ~1658.4 cm−1 in the

343

FT-IR spectrum (Figure 1B). Additionally, the average molecular weight of the

344

acid-labile PEI was determined to be 28.5 kDa by GPC.

345

13

C NMR

The accumulation of non-biodegradable polymers may be a serious problem in

16


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346

vivo. Simulating the pH of extracellular environment, endosomes and lysosomes, the

347

degradability of acid-labile PEI was examined in Figure 1C. The half-life of the

348

polymer degradation was 0.9 h, 3.6 h, and 129 h, respectively, at the corresponding

349

pH condition of 4.5, 5.4 and 7.4. The buffering capacity of polymeric gene delivery

350

system is closely related to the transfection efficiency. The high buffering capacity of

351

PEI ascribes to the high content of amino groups, thus facilitating endolysosomal

352

escape of nucleic acids. As seen in Figure 1D, acid-labile PEI exhibited high buffering

353

capacity, even similar with 25 kDa PEI. It is generally accepted that the cytotoxicity

354

and transfection efficiency of PEI are positively correlated with the molecular weight.

355

Higher molecular weight PEI (i.e. 25 kDa) has significant cytotoxicity and high

356

transfection efficiency, while lower molecular weight PEI (i.e. 1.8 kDa) shows low

357

transfection efficiency but negligible cytotoxicity.38 Therefore, crosslinking low

358

molecular weight PEI into high molecular weight PEI with degradable linkers may be

359

an alternative strategy to overcome the high cytotoxicity and mediate efficient gene

360

transfection.

361 362

Scheme 2. The synthesis of acid-labile PEI.

363

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364 365

Figure 1. Characterization of the acid-labile PEI in (A) 13C NMR and (B) FT-IR. (C)

366

Degradation of the acid-labile PEI in different pH conditions. (D) pH buffering

367

capacity of the acid-labile PEI, 25k PEI and 1.8k PEI.

368

3.2. Preparation and Characterization of acid-labile PEI/HNP

369

Before encapsulating anti-miR155 inside the core of HNP, acid-labile PEI was

370

first used to condense the anti-miR155. Agarose gel electrophoresis was performed to

371

investigate whether anti-miR155 molecules were retarded completely by acid-labile

372

PEIs. When the N/P ratio reached 5/1, almost all RNA molecules were complexed and

373

there was no free anti-miR155 band observable in Figure 3A. Dynamic light

374

scattering measurement revealed the changes of particle size at various N/P ratios in

375

Figure 2B, N/P ratio of 5/1 with the minimum size (179.2 ± 1.6 nm) was chosen as the

376

positively charged core (31.75 ± 1.69 mV) for the next step. The core was then

377

packaged in a lipid bilayer by thin-film hydration method, followed by extrusion to

378

apply a mechanical energy. After build-up of the lipid shell on the core (191.2 ± 0.8

379

nm), the zeta potential shifted from positive values to negative values (-24.87 ± 1.37

380

mV), indicating that the lipid bilayer was coated. Afterwards, apo A1 was assembled

18


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381

to form the final HNP by sodium cholate dialysis method with the diameter of 180.6 ±

382

0.4 nm and the zeta potential of -37.44 ± 1.63 mV. Fluorescent quantitative analysis of

383

acid-labile PEI/HNP revealed that the amount of anti-miR155 and apo A1 were

384

respectively 24000 nM and 103.5 nM.

385

The morphological characteristics of different nanoparticles were directly

386

visualized by TEM as illustrated in Figure 2A. The image of acid-labile PEI core

387

demonstrated the spherical shape formation. Acid-labile PEI/HNP appeared as a black

388

core surrounded by a rim, confirming the expected core-shell type HNP.

389 390

Figure 2. (A) TEM images of the acid-labile PEI core and acid-labile PEI/HNP. (B)

391

Changes of particle size and zeta potential of acid-labile PEI/anti-miRNA155

392

complexes at different N/P ratios. (C) Particle size and zeta potential of acid-labile

393

PEI core, acid-labile PEI/lipo and acid-labile PEI/HNP.

394

3.3. Serum Stability and RNase Protection Assay

395

When gene delivery vectors enter into the blood circulation, the nonspecific

396

interactions of vectors with serum components will influence the transfection

397

efficiency. Gel retardation assay was carried out to confirm the serum stability of

398

acid-labile PEI/HNP. Figure 3B is the electrophoregram incubated in 10% and 50%

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399

FBS, respectively. It can be seen that the bands of naked anti-miR155 gradually

400

blurred with time in 10% FBS and disappeared completely after 24 h in 50% FBS.

401

However, when anti-miR155 was incorporated into the HNP, there was almost no

402

degradation in both 10% and 50% FBS during 48 h, indicating that HNP protected

403

nucleic acids from the degradation and loss in circulatory system.

404

Another important factor affecting the efficient gene delivery is the enzymatic

405

degradation in vitro and in vivo. The RNase protection ability of vectors was

406

examined in Figure 3C. Naked anti-miR155 was quickly degraded when exposed to

407

the RNase A. In comparison with naked anti-miR155, the released anti-miR bands in

408

HNP were clear and intact. The result implied that protecting anti-miRNAs against

409

nucleases by desirable delivery vectors is necessary.

410 411

Figure 3. (A) Gel retardation assay of acid-labile PEI/anti-miR155 complexes at

412

various N/P ratios. (B) Serum stability test of (a) naked anti-miR155 and (b)

413

acid-labile PEI/HNP in 10% and 50% FBS. (C) Gel electrophoresis of naked

414

anti-miR155 and acid-labile PEI/HNP incubated for different periods with RNase A.

20


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415

3.4. Cell Viability Assay

416

The safety of materials is one of an important indicator for ideal gene vectors. In

417

order to compare the cytotoxicity of acid-labile PEI, 1.8 kDa PEI and 25 kDa PEI,

418

three polymers were complexed with anti-miR155 at similar particle size and zeta

419

potential and MTT assay was performed at different polymers concentration. The

420

results in Figure 4A showed that the cell viability of acid-labile PEI and 1.8 kDa PEI

421

remained unchanged as the polymer concentration increasing. However, the cells

422

treated with 25 kDa PEI had significantly higher cytotoxicity compared to those

423

treated with acid-labile PEI and 1.8 kDa PEI, and the viability dropped dramatically

424

to 6% when the polymer concentration reached 50 µg/mL. Liu et al.41 showed that

425

mixing PEI with negatively charged nucleic acids could shield or neutralize the

426

positive charges of the PEI molecules, thus lowering part of the cytotoxicity.

427

Nevertheless, when nucleic acids are released in cytoplasm after internalization, free

428

PEIs will interact with cellular components and disturb the normal cellular

429

process,38,42 especially for high molecular weight PEI. A major benefit of this

430

acid-labile PEI is that it can be degraded into low molecular weight PEI, which is

431

almost non-toxic to cells. Therefore, we expected that the acid-labile PEI could

432

substitute the 25 kDa PEI with the characteristics of high transfection while

433

maintaining the low cytotoxicity. Besides, after loading the cationic core inside the

434

HNP, the cell viabilities of various nanoparticles at tested concentrations were also

435

evaluated in Figure 4B, and no obvious cytotoxicity was observed.

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436 437

Figure 4. (A) Cell viability of three PEI core at various polymer concentrations (1-50

438

µg/mL). (B) Cell viability of different nanoparticles at an anti-miR155 concentration

439

of 200 nM.

440

3.5. Cellular Uptake Study and Active Targeting of acid-labile PEI/HNP to SR-B1

441

The uptake of different complexes was determined by using FAM labled

442

anti-miR155. As illustrated in Figure 5A and B, naked anti-miR155 was hardly

443

uptaked, suggesting that only with the help of vectors could the uptake be improved.

444

Acid-labile PEI core and 25 kDa PEI core had similar fluorescence signals which

445

were stronger than those of 1.8 kDa PEI core. Compared to acid-labile PEI core,

446

acid-labile PEI/lipo showed increased uptake which might be attributed to the polar

447

lipid bilayer facilitating the fusion with the cell membrane. Moreover, after modifying

448

with apo A1, the uptake of acid-labile PEI/HNP was about 2.1-fold higher than that of

449

non-targeted acid-labile PEI/lipo and about 3.2-fold higher than that of cationic

450

acid-labile PEI core in Raw 264.7 cells. To further confirm the role of SR-B1 in

451

mediating the uptake of acid-labile PEI/HNP, we also treated cells with the BLT-1, a

452

selective inhibitor of SR-B1. As exhibited in Figure 6, acid-labile PEI/HNP uptake

453

was decreased by 68.8% after the knock down of SR-B1 in Raw 264.7 cells.

454

Conversely, the uptake of acid-labile PEI core and acid-labile PEI/lipo in cells showed

455

no significant difference with BLT-1 treatment. The results demonstrated the active 22


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456

targeting of the acid-labile PEI/HNP to SR-B1 in macrophages.

457 458

Figure 5. (A) and (B) Quantitative analysis the cellular uptake behaviors of different

459

nanoparticles. (C) Influence of different inhibitors on the cellular uptake pathway of

460

acid-labile PEI/HNP.

461 462

Figure 6. (A) and (B) Inhibitory effect of BLT-1 on uptake of acid-labile PEI core (a),

463

acid-labile PEI/lipo (b) and acid-labile PEI/HNP (c) by Raw 264.7 cells.

464

3.6. Mechanism of Cellular Uptake

465

3.6.1. Cellular Trafficking Pathway

466

The endocytosis of nanoparticle is an active and energy-dependent process, which

467

can be inhibited by low temperature as well as ATP enzyme inhibitors.43 Therefore,

468

the two factors are usually the most direct way to distinguish the endocytic pathway

469

and the non-endocytic pathway. Sodium azide (NaN3) which can inhibit the

23


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470

production of energy by blocking the respiratory chain of mitochondria is widely used

471

as the inhibitor of energy dependence.43 As seen in Figure 5C, a decreasing cellular

472

uptake was observed in cells incubated at 4 °C or incubated with NaN3, clarifying the

473

energy-dependent endocytosis.

474

Generally, there are four types of endocytic pathway, including clathrin-mediated

475

endocytosis, caveolae-mediated endocytosis, macropinocytosis and phagocytosis.44 To

476

elucidate the cellular trafficking pathway of acid-labile PEI/HNP, different inhibitors

477

blocking specific pathway were used. Chlorpromazine hydrochloride prevents

478

clathrin-coated pit formation by promoting clathrin agglutination in late endosomes.45

479

In cells treated with chlorpromazine hydrochloride, there was a significant decrease

480

(68% reduction) in cellular uptake relative to the control. Genistein, a known inhibitor

481

of caveolae-mediated endocytosis, showed a considerably smaller impact on the

482

internalization in Raw 264.7 cell (17.6% reduction). Next, inhibitor of sodium-proton

483

exchange “amiloride” for the macropinocytosis inhibition44 exhibited a 38% reduction

484

in Raw 264.7 cell. It could be concluded that clathrin-mediated endocytosis was the

485

main uptake pathway for acid-labile PEI/HNP, and macropinocytosis was also an

486

effective uptake pathway in Raw 264.7 cells.

487

3.6.2. Intracellular Distribution

488

To further illuminate the trafficking mechanism of acid-labile PEI/HNP, the

489

intracellular behavior was investigated by confocal microscopy. FAM labeled

490

anti-miR155 was used to trace the nanoparticles, and the nucleus and acidic

491

endolysosomes were stained by Hoechst 33342 and Lyso-Tracker Red, respectively.

24


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Biomacromolecules

492

In Raw 264.7 cells, acid-labile/HNPs were observed bound to the cell membrane at 2

493

h (Figure 7). By 6 h post-treatment, intracellular green fluorescence began to appear

494

in larger and brighter compartments, suggestive of vesicles containing numerous

495

nanoparticles. Futhermore, the yellow fluorescence appeared in the cells marginally

496

which was the co-localization of FAM-anti-miR155 and Lyso-Tracker, declaring that

497

some nanoparticles had located in endolysosomes. After 16 h incubation, an increased

498

yellow fluorescence was visible in cells, indicating that most of the nanoparticles had

499

been entrapped within endolysosomes. This result was consistent with the conclusion

500

above that clathrin-mediated endocytosis was the primary endocytosis pathway. At the

501

time of 34 h, the yellow fluorescence of the image was weakened accompanied by the

502

appearance of large green fluorescence. It meant that the anti-miR155 had escaped

503

from the endolysosomes into cytoplasm successfully, mainly attributing to the high

504

buffering capacity of acid-labile PEI.

505

It is noteworthy that the intracellular trafficking and uptake mechanism of HDL

506

are dependent on different cell lines. For instance, the interaction of HDL with

507

hepatocytes involves the selective lipid uptake mediated by SR-B146 and the

508

holo-particle uptake mediated by SR-B1, CD36 and the mitochondrial β-chain of

509

ATP synthase and P2Y13.47,48 Yang and other researchers utilized the selective lipid

510

uptake pathway for HNP delivering nucleic acids directly into the cytoplasm thereby

511

bypassing the endosomal trapping.12-14 Researchers also took advantage of the

512

holo-particle uptake pathway of HNP for the target-specific delivery of siRNA.49

513

However, there has been no report about the selective lipid uptake pathway in

25


Biomacromolecules

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514

macrophages, which might be associated with the physiological functions of different

515

cells. In hepatocytes, mature HDL can unload free and esterified cholesterol by open a

516

non-aqueous channel in SR-B1, namely selective cholesteryl ester uptake pathway for

517

depletion.48,50 In contrast, HDL is responsible for mediating cholesterol efflux in

518

macrophages and foam cells. And in our experiments, FAM-anti-miR155 and

519

Cy5.5-apo A1 were used to label the acid-labile/HNP for characterizing the uptake

520

pathway. As shown in Figure S1, green and red fluorescences were located within the

521

cytosolic compartments and observed in the same positions at 6 h, demonstrating a

522

holo-particle uptake pathway of HNP in macrophages.

523 524

Figure 7. Confocal microscopy shows intracellular distribution of acid-labile

525

PEI/HNP. Green fluorescence represents the FAM-anti-miR155. Red fluorescence is

526

from Lyso-Tracker Red endolysosomes staining. Blue fluorescence is from the

527

nucleus stained with Hoechst 33342. Yellow fluorescence is from the co-localization

528

of green and red.

529

3.7. In vitro Gene Silencing Efficiency

530

Figure 8 shows the miR155 expression levels in different formulations containing 26


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531

anti-miR155 relative to the untreated group examined by qRT-PCR. Naked

532

anti-miR155 could hardly penetrate the cell membrane, resulting in low transfection

533

efficiency. Therefore, the acid-labile PEI as a vector with low cytotoxicity and high

534

transfection efficiency was synthesized. Packaging anti-miR155 into 1.8 kDa PEI and

535

25 kDa PEI formulations were used as the negative control and positive control

536

groups, respectively. As expected, acid-labile PEI and 25 kDa PEI treated groups

537

exhibited a similar and significantly reduced expression of miR155 levels compared

538

to the untreated group. There was almost no silencing efficiency in 1.8 kDa PEI

539

treated group. The high transfection efficiency of acid-labile PEI and 25 kDa PEI

540

formulations was attributed to the high charge density and the high buffering capacity,

541

thus triggering anti-miR155 escape from endolysosomes. Subsequently, the

542

transfection efficiency in each polymer treated group was compared. Treatment with

543

the acid-labile PEI or 25 kDa PEI/lipo caused a slight reduction in the expression of

544

miR155 levels to those of acid-labile PEI or 25 kDa PEI core. The positive surface

545

charges of acid-labile PEI or 25 kDa PEI core and the polar lipid bilayer of acid-labile

546

PEI or 25 kDa PEI/lipo were favorable for the cellular uptake, which contributed to

547

the increased gene silencing efficiency. In acid-labile PEI or 25 kDa PEI/HNP treated

548

group, the miR155 expression was strongly inhibited compared to that in acid-labile

549

PEI or 25 kDa PEI/lipo treated group, and the reduction was 22% and 23.5%

550

respectively, exhibiting the advantage of specific targeting of HNP.

551

27


Biomacromolecules

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552 553

Figure 8. In vitro gene silencing efficiency after the transfection with different

554

nanoparticles at an anti-miR155 concentration of 200 nM.

555

3.8. DiI-oxLDL Uptake

556

Lipid uptake plays a central role in macrophage-derived foam cell formation.51

557

Tian proved that miR155 promoted lipid uptake and ROS production to enchance

558

macrophages-derived foam cell formation in vitro via direct suppression of HMG

559

box-transcription protein1 (HBP1). Conversely, inhibition of miR155 reduced lipid

560

uptake at a dose dependent manner.7 In order to explore the efficacy of various

561

formulations containing anti-miR155, DiI-oxLDL uptake assay was performed to

562

evaluate the intracellular lipid contents (Figure 9). Compared to the positive control

563

group, there was no obvious inhibition on lipid uptake in the treatment of naked

564

anti-miR155 alone. When the anti-miR155 was packaged in different formulations,

565

the lipid uptake gradually decreased in the order of acid-labile PEI core, acid-labile

566

PEI/lipo and acid-labile PEI/HNP group. Excluding the effect of anti-miR155, the

567

blank HNP also exhibited the function of inhibiting lipid uptake in acid-labile

568

PEI/HNP loaded with negative control anti-miRNA relative to that in acid-labile PEI

569

core N.C. and acid-labile PEI/lipo N.C. groups. Cho et al. also demonstrated that 28


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Biomacromolecules

570

rHDL treatment had the antioxidant activity by inhibiting cellular uptake of oxLDL.19

571

Furthermore, to exclude a potential competition between ox-LDL and HNP,

572

incubating acid-labile PEI/HNP N.C. with Raw 254.7 cells, with or without washing

573

before stimulated with oxLDL was performed to make a comparison. There was no

574

significant difference in the uptake of DiI-oxLDL whether there was a washing after

575

the treatment of acid-labile PEI/HNP N.C. (Figure S2), meaning that the reduction of

576

DiI-oxLDL uptake was not caused by the competition of the HNP. Above all, these

577

findings suggested that both anti-miR155 and blank HNP inhibited the

578

oxLDL-induced foam cell formation.

579

Recent studies have revealed the potential anti-atherosclerotic effect of miR155

580

inhibition. MiR155 deficiency in apo E-deficient mice resulted in decreased

581

macrophage inflammations and lipid dispositions and attenuated atherogenesis by

582

interfering with different pathway.6-8 However, others reported that LDL receptor

583

deficiency (LDLR−/−) mice with miR155−/− bone marrow cells showed a contradictory

584

outcome with enchanced atherosclerosis.52 The inconsistent results are speculated to

585

be dependent on different animal models of the disease. The detailed atheroprotection

586

of anti-miR155 in our experiments needs to be further verified in atherosclerotic

587

animal models.

588 589

Figure 9. DiI-oxLDL uptake images captured by fluorescence microscope.

29


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590

3.9. ROS Production

591

ROS production is another important factor effects foam cell formation, which

592

has been proved that transfection of miR155 in Raw 264.7 cells enhanced ROS

593

production, while inhibition of miR155 presented the opposite effects.7 As shown in

594

Figure 10A, exposure of cells to formulations containing anti-miR155 led to a marked

595

decrease in ROS production compared to the blank cells only irritated with oxLDL.

596

The production of ROS in various formulations decreased in the following order:

597

acid-labile PEI core group, acid-labile PEI/lipo group and acid-labile PEI/HNP group,

598

respectively. We next examined whether blank HNP affects ROS generation.

599

Interestingly, acid-labile PEI/HNP N.C. showed a significantly suppression of ROS

600

generation relative to the positive control. Tölle reported that HDL had the capacity to

601

inhibit NAD(P)H oxidase-dependent ROS generation in vascular smooth muscle

602

cells.53 In Figure S3, the production of ROS also displayed no obvious difference with

603

or without washing after the treatment of acid-labile PEI/HNP N.C., eliminating the

604

potential competition between oxLDL and HNP. It is hoped that this prepared

605

acid-labile PEI/HNP can not only play the role of the anti-miR155, but also exert the

606

biological function of the HNP itself.

607 608

Figure 10. (A) ROS production and (B) Cholesterol efflux assay in Raw 264.7 cells

609

after the incubation with different nanoparticles. 30


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Biomacromolecules

610

3.10. Cholesterol Efflux Assay

611

It has been well known that cholesterol efflux from macrophages to HDL

612

represents an important mechanism for maintaining cholesterol homeostasis. To

613

examine whether miR155 and HNP have the capacity of mediating cholesterol efflux,

614

NBD-cholesterol and ox-LDL were pre-incubated with Raw 264.7 cells. As seen in

615

Figure 10B, there was a small increase of cholesterol efflux in the presence of

616

acid-labile PEI/lipo and acid-labile PEI core compared to the control and naked

617

anti-miR155 group, whereas acid-labile PEI/HNP group displayed significantly

618

increased cholesterol efflux. Moreover, we found that cholesterol efflux to blank HNP

619

was significantly increased compared to the control, but efflux to acid-labile PEI/lipo

620

N.C. and acid-labile PEI core N.C. was not changed, manifesting the potential RCT

621

function of HNP. It was reported that SR-B1-mediated HDL endocytosis leads to the

622

resecretion of HDL.48,54 During the retro-endocytosis, HDL interacted with lipid

623

droplets in foam cells for the exchange of cholesterol to HDL, thus facilitating

624

cholesterol efflux. Besides, we speculated that the lipids bilayer structure also

625

provided a space for sequestering more cholesterol as demonstrated by Luthi.35

626

CONCLUSIONS

627

In summary, a biomimetic polymer-lipid hybrid HNP loading anti-miR155 was

628

established successfully for combined antiatherogenic effects on macrophages with

629

nanocarrier and nucleic acid. The developed HNP realized the macrophages-specific

630

targeting and escaped from the endolysosomes via clathrin-mediated endocytosis with

631

high transfection efficiency. Besides, in vitro cells confirmed that the HNP closely

31


Biomacromolecules

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

632

mimicking endogenous HDL in the unique anti-atherosclerotic biological function had

633

the ability of antioxidation and mediating cholesterol efflux. Further in vivo

634

pharmacodynamics of both anti-miR155 and nanocarrier are still required to be

635

carried out in atherosclerotic animal models.

636

ASSOCIATED CONTENT

637

Supporting Information

638

Confocal images of acid-labile PEI/HNP, DiI-oxLDL uptake images of acid-labile

639

PEI/HNP N.C. and the ROS production of acid-labile PEI/HNP N.C..

640

AUTHOR INFORMATION

641

Corresponding Authors

642

*E-mail: [email protected]. Fax：+86-25-83271293

643

Notes

644

The authors declare no competing financial interests.

645

ACKNOWLEDGMENTS

646

The work was financially supported by National Natural Science Foundation of China

647

Project (No. 81273466) and Priority Academic Program Development of Jiangsu

648

Higher Education Institutions. We also thank you Minhui Sun, Yingjian Hou, and

649

Xiaonan Ma, for technical support from Cellular and Molecular Biology Center of

650

China Pharmaceutical University.

651

REFERENCES

652

(1) Weber, C.; Noels H. Atherosclerosis: current pathogenesis and therapeutic

653

options. Nat. Med. 2011, 17, 1410-1422.

32


Page 32 of 54

Page 33 of 54

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

Biomacromolecules

654

(2) Moore, K. J.; Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell

655

2011, 145, 341-355.

656

(3) Li, A. C.; Glass, C. K. The macrophage foam cell as a target for therapeutic

657

intervention. Nat. Med. 2002, 8, 1235-1243.

658

(4) Madrigal-Matute, J.; Rotllan, N.; Aranda, J. F.; Fern á ndez-Hernando, C.

659

MicroRNAs and atherosclerosis. Curr. Atheroscler. Rep. 2013, 15, 1-8.

660

(5) Condorelli, G.; Latronico, M. V. G.; Cavarretta, E. MicroRNAs in cardiovascular

661

diseases: current knowledge and the road ahead. J. Am. Coll. Cardiol. 2014, 63,

662

2177-2187.

663

(6) Nazari-Jahantigh, M.; Wei, Y.; Noels, H.; Akhtar, S.; Zhou, Z.; R.Koenen, R.; Heyll,

664

K.; Gremse, F.; Kiessling, F.; Grommes, J.; Weber, C.; Schober, A. MicroRNA-155

665

promotes atherosclerosis by repressing Bcl6 in macrophages. J. Clin. Invest. 2012,

666

122, 4190-4202.

667

(7) Tian, F. J.; An, L. N.; Wang, G. K.; Zhu, J. Q.; Zhang, Y. Y.; Zeng, A.; Zou, J.;

668

Zhu, R. F.; Han, X. S.; Shen, N.; Yang, H. T.; Zhao, X. X.; Huang, S.; Qin, Y. W.;

669

Jing, Q. Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in

670

atherogenesis. Cardiovasc. Res. 2014, 103, 100-110.

671

(8) Du, F.; Yu, F.; Wang, Y.; Hui, Y.; Carnevale, K.; Fu, M.; Lu, H.; Fan, D.

672

MicroRNA-155 deficiency results in deceased macrophage inflammation and

673

attenuated atherogenesis in apolipoprotein E-deficient Mice. Arterioscl. Throm. Vas.

674

2014, ATVBAHA. 113.302701.

33


Biomacromolecules

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

675

(9) Singh, A.; Talekar, M.; Raikar, A.; Aimiji, M. Macrophage-targeted delivery

676

systems for nucleic acid therapy of inflammatory diseases. J. Controlled Release

677

2014, 190, 515-530.

678

(10) Damiano, M. G.; Mutharasan, R. K.; Tripathy, S.; McMahon, K. M.; Thaxton, C.

679

S. Templated high density lipoprotein nanoparticles as potential therapies and for

680

molecular delivery. Adv. Drug Delivery Rev. 2013, 65, 649-662.

681

(11) McMahon, K. M.; Mutharasan, R. K.; Tripathy, S.; Veliceasa, D.; Bobeica, M.;

682

Shumaker, D.K.; Luthi, A. J.; Helfand,B. T.; Ardehail, H.; Mirkin, C. A.; Volpert, D.;

683

Thaxton, C. S. Biomimetic high density lipoprotein nanoparticles for nucleic acid

684

delivery. Nano. Lett. 2011, 11, 1208-1214.

685

(12) Yang, M.; Jin, H.;Chen, J.; Ding, L.; Ng, K. K.; Lin, Q. Y.; Lovell, J. F.; Zhang,

686

Z. H.; Zheng, G. Efficient cytosolic delivery of siRNA using HDL‐mimicking

687

nanoparticles. Small 2011, 7, 568-573.

688

(13) Lin, Q.; Chen, J.; Jin, H.; Ng, K. K.; Yang, M.; Cao, W. G.; Ding, L. L.; Zhang,

689

Z. H.; Zheng, G. Efficient systemic delivery of siRNA by using high-density

690

lipoprotein-mimicking peptide lipid nanoparticles. Nanomedicine 2012, 7, 1813-1825.

691

(14) Zhang, Z.; Cao, W.; Jin, H.; Lovell, J. F.; Yang, M.; Ding, L. L.; Chen, J.;

692

Corbin, L.; Luo, Q. M.; Zheng, G. Biomimetic nanocarrier for direct cytosolic drug

693

delivery. Angew. Chem. Int. Edit. 2009, 48, 9171-9175.

694

(15) McMahon, K. M.; Plebanek, M. P.; Thaxton, C. S. Properties of Native High‐

695

Density Lipoproteins Inspire Synthesis of Actively Targeted In Vivo siRNA Delivery

696

Vehicles. Adv. Funct. Mater. 2016, 26, 7824-7835.

34


Page 34 of 54

Page 35 of 54

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

Biomacromolecules

697

(16) Shahzad, M. M. K.; Mangala, L. S.; Han, H. D.; Lu, C. H.; Bottsford-Miller, J.;

698

Nishimura, M.; Mora, E. M.; Lee, J. W.; Stone, R. L. Targeted delivery of small

699

interfering RNA using reconstituted high-density lipoprotein nanoparticles. Neoplasia

700

2011, 13, 309IN3-319IN8.

701

(17) Krause, B. R.; Remaley, A. T. Reconstituted HDL for the acute treatment of

702

acute coronary syndrome. Curr. Opin. Lipidol. 2013, 24, 480-486.

703

(18) Newton, R. S.; Krause, B. R. HDL therapy for the acute treatment of

704

atherosclerosis. Atherosclerosis Sup. 2002, 3, 31-38.

705

(19) Cho, K. H.; Kim, J. R. A reconstituted HDL containing V156K or R173C apoA-I

706

exhibited anti-inflammatory activity in apo-E deficient mice and showed resistance to

707

myeloperoxidase-mediated oxidation. Exp. Mol. Med. 2009, 41, 417-428.

708

(20) Badimon, J. J.; Badimon, L.; Galvez, A.; Dische, R.; Fuster, V. High density

709

lipoprotein plasma fractions inhibit aortic fatty streaks in cholesterol-fed rabbits.

710

Scand. J. Clin. Lab. Inv. 1989, 60, 455-461.

711

(21) Badimon, J. J.; Badimon, L,; Fuster, V. Regression of atherosclerotic lesions by

712

high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J. Cin. Invest.

713

1990, 85, 1234.

714

(22) Tardif, J. C.; Grégoire, J.; L’Allier, P. L.; Ibrahim, R.; Lespérance, J.; Heinonen,

715

T. M.; Kouz, S.; Berry, C.; Basser, R.; Lavoie, M. A.; Guertin, M. C.; Rodes-Cabau,

716

J. Effects of reconstituted high-density lipoprotein infusions on coronary

717

atherosclerosis: a randomized controlled trial. Jama 2007, 297, 1675-1682.

35


Biomacromolecules

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

718

(23) Shaw, J. A.; Bobik, A.; Murphy, A.; Kanellakis, P.; Blombery, P.;

719

Mukhamedova, N.; Woollard, K.; Lyon, S.; Sviridov, D.; Dart, A. M. Infusion of

720

reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic

721

plaque. Circ. Res. 2008, 103, 1084-1091.

722

(24) Orekhov, A. N.; Misharin, A. Y.; Tertov, V. V.; Khashimov, K.; Pokrovsky, S.

723

N.; Repin, V. S.; Smirnov, V. N. Artificial HDL as an anti-atherosclerotic drug.

724

Lancet 1984, 324, 1149-1150.

725

(25) Koizumi, J.; Kano, M.; Okabayashi, K.; Jadhav, A.; Thompson, G. R. Behavior

726

of human apolipoprotein AI: phospholipid and apoHDL: phospholipid complexes in

727

vitro and after injection into rabbits. J. Lipid. Res. 1988, 29, 1405-1415.

728

(26) Kontush, A.; Chapman, M. J. Antiatherogenic function of HDL particle

729

subpopulations: focus on antioxidative activities. Curr. Opin. Lipidol. 2010, 21,

730

312-318.

731

(27) Kontush, A.; Chapman, M. J. Antiatherogenic small, dense HDL-guardian angel

732

of the arterial wall. Nat. Clin. Pract. Card. 2006, 3, 144-153.

733

(28) Cockerill, G. W.; Rye, K. A.; Gamble, J. R.; Vadas, M. A.; Barter, P. J.

734

High-density lipoproteins inhibit cytokine-induced expression of endothelial cell

735

adhesion molecules. Arterioscl. Throm. Vas. 1995, 15, 1987-1994.

736

(29) Ansell, B. J.; Navab, M.; Hama, S.; Kamranpour, N.; Fonarow, G.; Hough, G.;

737

Rahmani, S.; Mottahedeh, R.; Dave, R.; Reddy, S. T.; Fogelman, A. M.

738

Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish

739

patients from control subjects better than high-density lipoprotein cholesterol levels

36


Page 36 of 54

Page 37 of 54

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

Biomacromolecules

740

and are favorably affected by simvastatin treatment. Circulation 2003, 108,

741

2751-2756.

742

(30) Sugano, M.; Tsuchida, K.; Makino, N. High-density lipoproteins protect

743

endothelial cells from tumor necrosis factor-α-induced apoptosis. Biochem. Bioph.

744

Res. Co. 2000, 272, 872-876.

745

(31) Nofer, J. R.; Van Der Giet, M.; Tölle, M.; Wolinska, I.; Lipinski, K. W.; Baba, H.

746

A.; Tietge, U. J.; Godecke, A.; Ishii, I.; Kleuser, B.; Schafers, M.; Fobker, M.; Zidek,

747

W.; Assmann, G.; Chun, J.; Levkau, B. HDL induces NO-dependent vasorelaxation

748

via the lysophospholipid receptor S1P 3. J. Clin. Invest. 2004, 113, 569-581.

749

(32) Yang, S; Damiano, M. G.; Zhang, H.; Tripathy, S.; Luthi, A. J.; Rink, J. S.;

750

Ugolkov, A. V.; Singh, A. T. k.; Dave, S. S.; Gordon, L. I.; Thaxton, C. S.

751

Biomimetic, synthetic HDL nanostructures for lymphoma. P. Natl. Acad. Sci. 2013,

752

110, 2511-2516.

753

(33) Numata, M.; Grinkova, Y. V.; Mitchell, J. R.; Chu, H. W.; Sligar, S. G.; Voelker,

754

D. R. Nanodiscs as a therapeutic delivery agent: inhibition of respiratory syncytial

755

virus infection in the lung. Int. J. Nanomed. 2013, 8, 1417-1427.

756

(34) Mutharasan, R. K.; Foit, L.; Thaxton, C. S. High-density lipoproteins for

757

therapeutic delivery systems. J. Mater. Chem. B 2016, 4, 188-197.

758

(35) Luthi, A. J.; Zhang, H.; Kim, D.; Giljohann, D. A.; Mirkin, C. A.; Thaxton, C. S.

759

Tailoring of biomimetic high-density lipoprotein nanostructures changes cholesterol

760

binding and efflux. ACS Nano 2011, 6, 276-285.

37


Biomacromolecules

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

Page 38 of 54

761

(36) Zhang, M. Y,; He, J. H.; Jiang, C.P.; Zhang, W. L.; Yang, Y.; Wang, Z. Y.; Liu, J.

762

P. Plaque-hyaluronidase-responsive high-density-lipoprotein-mimetic nanoparticles

763

for

764

anti-atherosclerotic therapy. Int. J. Nanomed. 2017, 12, 533.

765

(37) Behr, J. P. The proton sponge: a trick to enter cells the viruses did not exploit.

766

CHIMIA International Journal for Chemistry. 1997, 51, 34-36.

767

(38) Kim, Y. H.; Park, J. H.; Lee, M.; Kim, Y. H.; Park, T. G.; Kim, S. W. Polyethylene

768

with acid-labile linkages as a biodegradable gene carrier. J. Controlled Release 2005,

769

103, 209-219.

770

(39) He, H.; Liu, L.; Bai, H.; Wang, J.; Zhang, Y.; Zhang, W. L.; Zhang, M. Y.; Wu,

771

Z.; Liu, J. P. Arachidonic acid-modified lovastatin discoidal reconstituted high density

772

lipoprotein markedly decreases the drug leakage during the remodeling behaviors

773

induced by lecithin cholesterol acyltransferase. Pharm. Res. 2014, 31, 1689-1709.

774

(40) Chen, S.; Jin, T. Poly-cross-linked PEI through aromatically conjugated imine

775

linkages as a new class of pH-responsive nucleic acids packing cationic polymers.

776

Front. Pharmacol. 2016, 7.

777

(41) Liu, Z.; Niu, D.; Zhang, J.; Zhang, W.; Yao, Y.; Li, P.; Gong, J. Amphiphilic

778

core-shell nanoparticles containing dense polyethyleneimine shells for efficient

779

delivery of microRNA to Kupffer cells. Int. J. Nanomed. 2016, 11, 2785.

780

(42) Godbey, W. T.; Wu, K. K.; Mikos, A. G. Poly (ethylenimine)-mediated gene

781

delivery affects endothelial cell function and viability. Biomaterials 2001, 22,

782

471-480.

multistage

intimal-macrophage-targeted

drug

delivery

38


and

enhanced

Page 39 of 54

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

Biomacromolecules

783

(43) Liu, P.; Sun, Y.; Wang, Q.; Sun, Y.; Li, H.; Duan, Y. Intracellular trafficking and

784

cellular uptake mechanism of mPEG-PLGA-PLL and mPEG-PLGA-PLL-Gal

785

nanoparticles for targeted delivery to hepatomas. Biomaterials 2014, 35, 760-770.

786

(44) Xiang, S.; Tong, H.; Shi, Q.; Fernandes, J. C.; Jin, T.; Dai, K.; Zhang, X. Uptake

787

mechanisms of non-viral gene delivery. J. Controlled Release 2012, 158, 371-378.

788

(45) Wang, L. H.; Rothberg, K. G.; Anderson, R. G. W. Mis-assembly of clathrin

789

lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol.

790

1993, 123, 1107-1107.

791

(46) Rodrigueza, W. V.; Thuahnai, S. T.; Temel, R. E.; Lund-Katz, S.; Phillips, M. C.;

792

Williams, D. L. Mechanism of scavenger receptor class B type I-mediated selective

793

uptake of cholesteryl esters from high density lipoprotein to adrenal cells. J. Biol.

794

Chem. 1999, 274, 20344-20350.

795

(47) Röhrl, C.; Pagler, T. A.; Strobl, W.; Ellinger, A.; Neumuller, J.; Pasvelka, M.;

796

Stangl, H.; Meisslitzer-Ruppitsch, C. Characterization of endocytic compartments

797

after holo-high density lipoprotein particle uptake in HepG2 cells. Histochem. Cell

798

Biol. 2010, 133, 261-272.

799

(48) Röhrl, C.; Stangl, H. HDL endocytosis and resecretion. Bba-mol. Cell Biol. L.

800

2013, 1831, 1626-1633.

801

(49) Rui, M.; Tang, H.; Li, Y.; Wei, X.; Xu, Y. Recombinant high density lipoprotein

802

nanoparticles for target-specific delivery of siRNA. Pharm. Res. 2013, 30, 1203-1214.

803

(50) Rodrigueza, W. V.; Thuahnai, S. T.; Temel, R. E.; Lund-Katz, S.; Phillips, M. C.;

804

Williams, D. L. Mechanism of scavenger receptor class B type I-mediated selective

39


Biomacromolecules

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

805

uptake of cholesteryl esters from high density lipoprotein to adrenal cells. J. Biol.

806

Chem. 1999, 274, 20344-20350.

807

(51) Li, A. C.; Glass, C. K. The macrophage foam cell as a target for therapeutic

808

intervention. Nat. Med. 2002, 8, 1235-1243.

809

(52) Donners, M. M. P. C.; Wolfs, I. M. J.; Stöger, L. J.; Vorst, E.; Pottgens, C.;

810

Heymans, S.; Schroen, B.; Gijbels, M.; Winher, M. Hematopoietic miR155 deficiency

811

enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice. Plos

812

One 2012, 7, e35877.

813

(53) Tölle, M.; Pawlak, A.; Schuchardt, M.; Kawamura, A.; Tietge, U. J.; Lorkowski,

814

S.; Keul, P.; Assmann, G.; Chun, J.; Levkau, B.; Giet, M.; Nofer, J. R. HDL-associated

815

lysosphingolipids inhibit NAD (P) H oxidase-dependent monocyte chemoattractant

816

protein-1 production. Arterioscl. Throm. Vas. 2008, 28, 1542-1548.

817

(54) Pagler, T. A.; Rhode, S.; Neuhofer, A.; Laggner, H.; Strobl, W.; Hinterndorfer, C.;

818

Volf, I.; Pavelka, M.; Eckhardt, E. R. M.; Westhuyzen, D. R. Schutz, G. J.; Stangl, H.

819

SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion

820

facilitating cholesterol efflux. J. Biol. Chem. 2006, 281, 11193-11204.

821 822 823 824 825 826

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827

For Table of Contents Use Only

831

Biofunctional polymer-lipid hybrid high density lipoprotein-mimicking nanoparticles loading anti-miR155 for combined antiatherogenic effects on macrophages

832

Jing Lu, Yi Zhao, Xiaoju Zhou, Jian Hua He, Yun Yang, Cuiping Jiang, Zitong Qi,

833

Wenli Zhang and Jianping Liu*

834

Department of Pharmaceutics, China Pharmaceutical University, Nanjing, PR China

828 829 830

835 836

Schematic illustration of the acid-labile PEI/HNP trafficking in Raw 264.7 cell.

41


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Schematic illustration of the acid-labile PEI/HNP trafficking in Raw 264.7 cell. 35x14mm (300 x 300 DPI)


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Figure 1. Characterization of the acid-labile PEI in (A) 13C NMR and (B) FT-IR. (C) Degradation of the acidlabile PEI in different pH conditions. (D) pH buffering capacity of the acid-labile PEI, 25k PEI and 1.8k PEI. 64x50mm (300 x 300 DPI)


Biomacromolecules

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

Figure 2. (A) TEM images of the acid-labile PEI core and acid-labile PEI/HNP. (B) Changes of particle size and zeta potential of acid-labile PEI/anti-miRNA155 complexes at different N/P ratios. (C) Particle size and zeta potential of acid-labile PEI core, acid-labile PEI/lipo and acid-labile PEI/HNP. 53x34mm (300 x 300 DPI)


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Figure 3. (A) Gel retardation assay of acid-labile PEI/anti-miR155 complexes at various N/P ratios. (B) Serum stability test of (a) naked anti-miR155 and (b) acid-labile PEI/HNP in 10% and 50% FBS. (C) Gel electrophoresis of naked anti-miR155 and acid-labile PEI/HNP incubated for different periods with RNase A. 74x73mm (300 x 300 DPI)


Biomacromolecules

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Figure 4. (A) Cell viability of three PEI core at various polymer concentrations (1-50 µg/mL). (B) Cell viability of different nanoparticles at an anti-miR155 concentration of 200 nM. 33x13mm (300 x 300 DPI)


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Figure 5. (A) and (B) Quantitative analysis the cellular uptake behaviors of different nanoparticles. (C) Influence of different inhibitors on the cellular uptake pathway of acid-labile PEI/HNP. 42x11mm (300 x 300 DPI)


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Figure 6. (A) and (B) Inhibitory effect of BLT-1 on uptake of acid-labile PEI core (a), acid-labile PEI/lipo (b) and acid-labile PEI/HNP (c) by Raw 264.7 cells. 61x45mm (300 x 300 DPI)


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Biomacromolecules

Figure 7. Confocal microscopy shows intracellular distribution of acid-labile PEI/HNP. Green fluorescence represents the FAM-anti-miR155. Red fluorescence is from Lyso-Tracker Red endolysosomes staining. Blue fluorescence is from the nucleus stained with Hoechst 33342. Yellow fluorescence is from the co-localization of green and red. 66x53mm (300 x 300 DPI)


Biomacromolecules

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Figure 8. In vitro gene silencing efficiency after the transfection with different nanoparticles at an antimiR155 concentration of 200 nM. 57x40mm (300 x 300 DPI)


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Biomacromolecules

Figure 9. DiI-oxLDL uptake images captured by fluorescence microscope. 31x12mm (300 x 300 DPI)


Biomacromolecules

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Figure 10. (A) ROS production and (B) Cholesterol efflux assay in Raw 264.7 cells after the incubation with different nanoparticles. 34x14mm (300 x 300 DPI)


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Biomacromolecules

Scheme 1. Schematics of the acid-labile PEI/HNP. 77x72mm (300 x 300 DPI)


Biomacromolecules

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Scheme 2. The synthesis of acid-labile PEI. 177x53mm (300 x 300 DPI)


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Biofunctional PolymerâLipid Hybrid High-Density Lipoprotein

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Biofunctional PolymerâLipid Hybrid High-Density Lipoprotein