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Nanostructured lipid carriers accumulate in atherosclerotic plaques of ApoE–/– mice

Jonathan Vigne, Claudia Cabella, László Dézsi, Emilie Rustique,

Anne-Claude Couffin, Rachida Aid, Nadège Anizan, Cédric Chauvierre, Didier Letourneur, Dominique Le Guludec, et al.

To cite this version:

Jonathan Vigne, Claudia Cabella, László Dézsi, Emilie Rustique, Anne-Claude Couffin, et al.. Nanos-

tructured lipid carriers accumulate in atherosclerotic plaques of ApoE–/– mice. Nanomedicine: Nan-

otechnology, Biology and Medicine, Elsevier, 2019, 25, pp.102157. �10.1016/j.nano.2020.102157�. �hal-

03041642�

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1

Graphical Abstract

2 Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2020) xxx–xxx

4

5

Nanostructured lipid carriers accumulate in atherosclerotic plaques of

6

ApoE

−/−

mice

7

8 Jonathan Vigne, PharmD, MSa,b,c, Claudia Cabella, PhDd, László Dézsi, PhDe, Emilie Rustique, MSf, Anne-Claude Couffin, PhDf, Rachida Aid, MSc, 9 Nadège Anizan, PhDc, Cédric Chauvierre, PhDa, Didier Letourneur, PhDa, Dominique Le Guludec, MD PhDa,b,c, François Rouzet, MD PhDa,b,c, 10 Fabien Hyafil, MD PhDa,b,c, Tamás Mészáros, PhDe, Tamás Fülöp, MSe, János Szebeni, MD PhDe, Alessia Cordaro, PhDd, Paolo Oliva, MSd, 11 Véronique Mourier, PhDf, Isabelle Texier, PhDf,

12

13 aUniversité de Paris, LVTS, INSERM U1148, Paris, France

14 bNuclear Medicine Department, X. Bichat Hospital, APHP and DHU FIRE, Paris, France 15 cUniversité de Paris, UMS34 FRIM, Paris, France

16 dCentro Ricerche Bracco, Bracco Imaging SpA, Colleretto Giacosa, Italy

17 eNanomedicine Research and Education Center, Institute of Pathophysiology, Semmelweis University, Budapest, Hungary 18 fUniversity Grenoble Alpes, CEA, LETI,Grenoble, France

19

20 Autoradiography of64Cu-labelled nanostructured lipid carriers (NLC) and Oil Red O histology (neutral lipids staining) of aortas showed significant particle uptake 21 in atherosclerotic lesions 24 h after injection in ApoE−/−mice atherosclerotic models. Produced by well-controlled and up-scalable high pressure homogenization, 22 NLC could present similar features than lipoproteins, and be used as synthetic mimetics to convey drugs and contrast agents to atherosclerotic lesions.

23 24 25

xx (xxxx) xxx

nanomedjournal.com

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1

2Q1

Nanostructured lipid carriers accumulate in atherosclerotic plaques of

3

ApoE −/− mice

4Q3

Jonathan Vigne, PharmD, MS a, b, c , Claudia Cabella, PhD d , László Dézsi, PhD e ,

5

Emilie Rustique, MS f , Anne-Claude Couffin, PhD f , Rachida Aid, MS c , Nadège Anizan, PhD c ,

6

Cédric Chauvierre, PhD a , Didier Letourneur, PhD a , Dominique Le Guludec, MD PhD a,b,c ,

7

François Rouzet, MD PhD a,b,c , Fabien Hyafil, MD PhD a,b,c , Tamás Mészáros, PhD e ,

8

Tamás Fülöp, MS e , János Szebeni, MD PhD e , Alessia Cordaro, PhD d , Paolo Oliva, MS d ,

9

Véronique Mourier, PhD f , Isabelle Texier, PhD f,

10 a

Université de Paris, LVTS, INSERM U1148, Paris, France

11 b

Nuclear Medicine Department, X. Bichat Hospital, APHP and DHU FIRE, Paris, France

12 c

Université de Paris, UMS34 FRIM, Paris, France

13 d

Centro Ricerche Bracco, Bracco Imaging SpA, Colleretto Giacosa, Italy

14 e

Nanomedicine Research and Education Center, Institute of Pathophysiology, Semmelweis University, Budapest, Hungary

15 f

University Grenoble Alpes, CEA, LETI, Grenoble, France

16

Revised 4 December 2019

17

Abstract

18

Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone

19

atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of

64

Cu-fonctionalized NLC in

20

atherosclerotic mice.

64

Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure

21

homogenization at tens-of-grams scale. 24 h after injection of

64

Cu-chelated particles in ApoE

−/−

mice, focal regions of the aorta showed

22

accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in

23

aortas isolated from ApoE

−/−

mice compared to wild type (WT) control (8.95 [7.58, 10.16] ×10

8

vs 4.59 [3.11, 5.03] ×10

8

QL/mm

2

, P b

24

0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical

25

translation potential for targeted delivery and imaging purposes in atherosclerosis.

26

© 2020 Published by Elsevier Inc.

27

Key words: Atherosclerosis; Nanostructured lipid carriers; PET/MR imaging; High pressure, homogenization; CARPA

28

Atherosclerosis is one of the world’s leading causes of vascular

29

diseases and still represents a major burden globally.

1

Atheroscle-

30

rotic lesions are mainly composed of cells (especially inflamma-

31

tory and immune cells), connective tissue elements, debris and

32

lipids within the arterial wall.

2

Advanced lesions contain abnormal

33

accumulation of lipoproteins and cholesterol esters (CE) with

34

concomitant chronic inflammation.

3

Atherosclerotic plaques

35

prone to rupture, also called vulnerable plaques, are usually

36

characterized by the presence of a lipid-rich necrotic core that is

37

covered by a thin fibrous cap and depleted of smooth muscle cells.

4 38

There is a need to develop strategies to manage plaque

39

treatment more effectively and establish precise diagnosis on

40

disease development status. Nanotechnology-based approaches

41

for targeted drug delivery have now entered clinical practice in the

42

field of oncology. Nanomedicine formulations have recently

43

demonstrated their interest to deliver efficiently active compounds

44

Nanomedicine: Nanotechnology, Biology, and Medicine

xx (xxxx) xxx

nanomedjournal.com

Funding statement: This work was supported by the EU (“NanoAthero”

project FP7-NMP-2012-LARGE-6-309820). The authors declare no conflict of interest.

Q2

Preliminary results were presented by Jonathan Vigne at the European Molecular Imaging Meeting, San Sebastian (Spain), 21st of March 2018. Authors have no interests to disclose.

Acknowledgments: Authors thank the Accelerator for Research in Radiochemistry and Oncology at Nantes Atlantic (ARRONAX, Nantes, France) for Copper-64 supply. Authors acknowledge the help of Marie Escudé (CEA-LETI) for particle preparation and characterization, Amandine Arnould (CEA-LITEN) for TEM characterization, Guillaume Even (INSERM U1148) for histology, François Scalbert and Joël Aerts (INSERM U1148) for imaging experiments, Mária Harvich-Velkei (Semmelweis University) for technical assistance in CARPA experiments. Imaging experiments were performed on a platform member of France Life Imaging network (grant ANR-11-INBS-0006). LETI/DTBS is part of the Arcane Labex program (grant ANR-12-LABX-003).

⁎ Corresponding author.

E-mail address: Isabelle.texier-nogues@cea.fr. (I. Texier).

https://doi.org/10.1016/j.nano.2020.102157 1549-9634/© 2020 Published by Elsevier Inc.

Please cite this article as: Vigne J., et al., Nanostructured lipid carriers accumulate in atherosclerotic plaques of ApoE

−/−

mice. Nanomedicine: NBM 2020;

xx:0-11, https://doi.org/10.1016/j.nano.2020.102157

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45

in atherosclerotic lesions.

5–11

Liposomes are the most employed

46

nanoplatform approach in clinical studies because they are easy to

47

prepare and customize, but new nanoparticles formulations are

48

emerging for this purpose.

7,8,12,13

Lipoprotein-based nanoparti-

49

cles, and in particular low density lipoproteins (LDL) and high

50

density lipoproteins (HDL), either in their native or recombinant

51

forms, constitute attractive nanoparticle carriers because of their

52

interactions with macrophages in atherosclerotic plaques.

14–16

53

Low density lipoproteins (LDL) loaded with dexamethasone were

54

shown to accumulate in atherosclerotic lesions and to reduce

55

significantly CE accumulation in the aorta of atherogenic mice.

17

56

The injection of reconstituted high density lipoprotein (rHDL)

57

loaded with statin in mice resulted in a decrease in the intensity of

58

inflammation in advanced atherosclerotic plaques.

18

59

Lipoproteins are intrinsically biocompatible, biodegradable,

60

poorly immunogenic, and enable natural targeting properties.

18,19

61

Nevertheless, their biogenic nature constitutes an important

62

challenge for reproducible and controlled manufacturing,

63

scaling-up process, cost, and translation to clinical studies.

64

Recombinant lipoprotein platforms assembled from synthetic

65

lipid emulsions and isolated apolipoproteins or peptide fragments

66

might represent an attractive alternate approach for the targeting of

67

atherosclerotic plaques.

16

68

Nanostructured lipid carriers (NLC) have aroused high interest as

69

drug delivery systems for the last 25 years.

20–22

NLC were found to

70

accumulate within lipid-recruiting tissues.

23

and some of these

71

display similar structural features than blood-circulating very low

72

density lipoproteins (VLDL) and chylomicrons, such as particle

73

diameter around 50-60 nm, lipid core composed of triglycerides

74

(C10-C18), and phospholipid-comprising shell.

16

However, they

75

remarkably differ on the shelf (i.e. before blood injection) by the

76

presence of PEGylated surfactants that confer the nanoparticles very

77

long term stability in ready-to-inject buffers, suitable for product

78

commercialization.

24

This PEGylated coating could be rapidly

79

screened in vivo, since previous studies have indicated that NLC are

80

rapidly covered by apolipoprotein corona after incubation in plasma. -

81 25

In addition, NLC can be produced easily and reproducibly by

82

High Pressure Homogenization (HPH), a process currently used in

83

food, cosmetics, and pharmaceutical industries.

26

84

Herein, full process and methodology for scaled-up produc-

85

tion and quality control of

64

Cu-labelled NLC were developed.

86

The efficiency of this new platform as a drug and contrast agent

87

carrier in a murine model of atherosclerosis was tested. As lipid

88

containing nanoparticles (e.g. liposomes, lipid complexes) could

89

cause hypersensitivity reactions (HSR), NLC safety properties

90

were monitored following the European Medicines Agency

91

(EMA) recommendations to ensure relevant quality of intrave-

92

nous liposomal innovator products,

27

especially using the

93

sensitive porcine complement activation-related pseudoallergy

94

(CARPA) model.

28–30

95

Methods

96

Materials

97

Suppocire NB™ was purchased from Gattefossé (Saint-

98

Priest, France), Lipoid ™ S75 (soybean lecithin at N75%

99

phosphatidylcholine) from Lipoid (Ludwigshafen, Germany),

Myrj ™ S40 (polyethylene glycol 40 stearate) and super-refined

100

soybean oil from Croda Uniqema (Chocques, France). B22280

101

chelate was synthetized as described in supplementary materials.

102

Copper-64 (t

1/2

= 12.7 h, β

+

; 17.8%, Eβ

+

max = 656 keV, β

,

103

38.4%, Eβ

max = 573 keV) was obtained from ARRONAX

104

cyclotron facility (GIP ARRONAX, Saint-Herblain, France)

105

using the reaction

64

Ni (p,n)

64

Cu and was delivered as

64

CuCl

2 106

in 0.1 M HCl. Product was filtered through sterile Acrodisc®

107

syringe filters (0.2 μm, 13 mm) with Supor® membrane (PALL

108

Corporation, USA) before use. Other chemicals were purchased

109

from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

110

Lipid nanoparticle synthesis

111

Lipid nanoparticles were prepared at the lab scale (750 mg of

112

particles) by ultrasonication similarly to previously described

113

protocols,

24,31

as well as by high pressure homogenization

114

(HPH) at larger scale (50 g of particles). For ultrasonication, the

115

lipid phase comprised 255 mg of soybean oil, 85 mg of

116

Suppocire ™ NB, 65 mg of lecithin, and 5 mg of amphiphilic

117

chelate B22280, and the aqueous phase 345 mg of Myrj ™ S40

118

and water (qsp 2 mL). For HPH, ingredient quantities were

119

scaled up 75 folds. Mixture of lipid and aqueous phases was pre-

120

emulsified using a mechanical disperser (Ultra-T25 Digital

121

Turrax, IKA) operated at 15,000 rpm for 5 min. The pre-

122

emulsion was processed with a High Pressure Homogenizer

123

(Panda Plus 2000, GEA Niro Soavi, Italy) operated for 16 cycles

124

with a total pressure of 1200 bars, the pressure of the second

125

stage chamber and the cooling system being set at 50 bars and 30

126

°C, respectively. 15 g of particles was purified by 5 μm filtration

127

followed by tangential flow filtration (Labscale TFF system,

128

Millipore) against 1 M acetate buffer pH 8.3 through a Pellicon

129

XL Biomax ™ cassette (Merck) operated at a trans-membrane

130

pressure of 14 psi at a flow rate of 2 mL/min. The nanoparticle

131

dispersion was adjusted to a concentration of 100 mg/mL and

132

filtered through a 0.22 μm Millipore membrane for sterilization

133

before storage and use.

134

Lipid nanoparticle characterizations

135

The endotoxin level in particle batches was measured by

136

Charles River (Ecully, France) using the chromogenic LAL test.

137

Dynamic light scattering (DLS) was used to determine the

138

particle hydrodynamic diameter and zeta potential (Zeta Sizer

139

Nano ZS, Malvern Instrument, Orsay, France). Particle disper-

140

sions were diluted to 2 mg/mL of lipids in 0.22 μm filtered 0.1×

141

PBS and transferred in Zeta Sizer Nano cells (Malvern

142

Instrument) before each measurement, performed in triplicate.

143

Results (Z-average diameter, dispersity index, zeta potential)

144

were expressed as mean and standard deviation of three

145

independent measurements performed at 25 °C. TEM images

146

of NLC were obtained after negative staining (2% uranyl acetate)

147

similarly to previously published protocol.

32 148

Radiolabeling procedures and analysis

149

150 μL of B22280-NLC (100 mg/mL of lipids) was incubated

150

with 200 ± 20 MBq of

64

CuCl

2

and ammonium acetate buffer (0.1

151

M, pH 7.0) in a rubber sealed vial. The mixture was heated at 60 °C

152

for 25 min then filtered through a 0.22 μm membrane (IC Acrodisc,

153

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154

13 mm syringe filter Supor® PES membrane, Pall Life Sciences).

155

Two controls were performed using the same protocol, first using

156

“nude” NLC and second, replacing B22280-NLC by ethylene-

157

diamine-tetra-acetic acid (EDTA, 5 mM).

158

Radiolabeling characterization was performed using PD-10

159

desalting columns prepacked with Sephadex™ G-25 Medium

160

(GE Healthcare, UK). Columns were equilibrated with saline,

161

then the sample containing the radiolabeled mixture (dissolved in

162

2.5 mL buffer) was added and the flow-through discarded.

163

Elution was performed with 5 mL saline by collecting eluate

164

fractions of 250 μ L. Each fraction was measured using a

64

Cu

165

calibrated activimeter MEDI404 (Medisystem, Guyancourt,

166

France). Radiochemical yield was measured using thin layer

167

chromatography (TLC) with a radiochromatograph (MiniGita,

168

Elysia-Raytest, Belgium). Stationary phase was silica gel (ITLC-

169

SG, Agilent technologies, CA, USA) and mobile phase was

170

0.068 mol/L citrate, 0.074 mol/L glucose, pH 5.0. Five days

171

post-radiolabeling, hydrodynamic diameter and dispersity index

172

were measured by DLS as previously described.

173

Complement activation assay

174

Five normal human sera activated by NLC or B22280-NLC

175

samples were measured with SC5b-9 EIA kit (TECOmedical AG,

176

Sissach, Switzerland). Positive complement activation control was

177

zymosan (0.3 mg/mL), whereas PBS was used as negative control.

178

One volume of sample (diluted with PBS to the desired

179

concentration) was added to 3 vol of sera. The mixture was gently

180

shaken for 45 min at 37 °C, then terminated by adding kit

181

Specimen Diluent supplemented with 10 mM EDTA. Samples

182

were frozen at −80 °C until SC5b-9 assay was performed, as

183

described in the kit instruction booklet. Optical density reading was

184

performed at 450 nm with a FLUOstar Omega plate reader (BMG

185

LABTECH GmbH, Offenburg, Germany).

186

CARPA test

187

CARPA studies in a porcine model were approved by the

188

National Scientific Ethical Committee on Animal Experimenta-

189

tion (ÁTET) and Institutional Animal Welfare Committee of

190

Semmelweis University (MÁB), and performed as previously

191

described.

28

Briefly, isoflurane-anesthetized Yorkshire pigs (20-

192

25 kg) were infused with saline (negative control) and chelated

193

or non-chelated NLC (Cu-B22280-NLC and B22280-NLC,

194

respectively) at 2.5 mL/min via the left external jugular vein at

195

doses of 7.5 and 37.5 (or 13.8) mg/kg, while 0.1 mg/kg zymosan

196

(positive control) was administered as a bolus injection. In one

197

experiment a bolus of non-chelated NLC was also applied.

198

Oxygen saturation, capnography, respiratory rate, pulmonary

199

arterial blood pressure (PAP), systemic arterial blood pressure

200

(SAP) and heart rate (HR) were continuously monitored.

201

Following blood sampling, plasma levels of thromboxane B2

202

(TXB2) were quantified using a commercially available ELISA

203

kit (Cayman Chemicals, Ann Arbor, MI, USA).

204

In vivo imaging experiments in mice

205

Animals

206

All procedures were performed in respect of the applicable

207

regulation for animal experimentation using protocols approved

by the animal care and use committee of the Claude Bernard

208

Institute (APAFIS #9681, Paris, France). ApoE

−/−

mice (in-

209

house breeding) aged over 35 weeks (weight 27-33 g) fed with

210

normal diet and wild type (WT) C57BL/6 mice (Charles River

211

laboratories) were housed under a 12 h light/dark cycle with free

212

access to water and normal chow ad libitum. Three experimental

213

groups were constituted: 1/ ApoE

−/−

group (n = 6) receiving

214 64

Cu-B22280-NLC (ApoE

−/−

group, experimental model of

215

atherosclerosis); 2/ WT group (n = 3) receiving

64

Cu-B22280-

216

NLC (WT control group) and 3/ ApoE

−/−

(n = 5) group receiving

217 64

CuCl

2

(ApoE

−/−

control group).

218

MicroPET/MRI & imaging data analysis

219

All PET/MRI scans were acquired using a Mediso NanoScan

220

PET/MRI (1 T Mediso Ltd., Budapest, Hungary). PET

221

acquisitions were performed 4 h and 24 h after retro-orbital

222

injection of the radiotracer (≈20 MBq ; 100-150 μL ; 1.5-2.25

223

mg NLC), followed by MRI acquisition (described in supple-

224

mentary materials). PET data were reconstructed using Tera-

225

Tomo™ 3D software (Mediso Ltd., Budapest, Hungary), a 3D-

226

OSEM Monte Carlo based algorithm with attenuation and scatter

227

corrections (voxel size equal to 0.3×0.3×0.3 mm

3

). Intensity

228

value in the voxels was calibrated in kilo Becquerel per cubic

229

centimeter (kBq cm

−3

) corrected for decay and positron

230

branching ratio of

64

Cu.

231

PET and MR images were automatically co-registered and

232

analyzed using PMOD software (v3.7, PMOD Technologies

233

Ltd). Volumes of interest (VOI

s

) were defined for the lungs,

234

bladder, right kidney, heart, muscle on the thigh, stomach and a

235

section of intestine on the MR images using threshold and

236

manual drawing, and for liver and spleen on the PET images

237

using threshold and manual drawing; blood activity was assessed

238

in the left ventricle using a 1.5 cm

3

ellipsoid volume. Percentage

239

of administered dose per gram of tissue (%ID/g) was calculated

240

for each organ using following equation:

241

%ID=g ¼ Mean activity in VOI kBq cm ð

−3

Þ =Organ density g cm ð

−3

Þ Injected activity kBq ð Þ

242 243244

The injected activity was corrected for decay and reference time was the PET acquisition.

245

Autoradiography, histology

246

Immediately after 24 h PET/MRI acquisition, mice were

247

euthanized with an overdose of isoflurane. Whole aortas were

248

collected from aortic root to bifurcation and washed with saline.

249

Aortas were dissected by removing adventitial adipose tissue

250

under binocular microscope and opened longitudinally to be lied

251

on slides. Radioactive signal on whole aortas was quantified

252

using a calibrated bio-image analyzer CR-35 Bio (Elysia-

253

Raytest, Liège, Belgium) after exposing samples for 24 h on

254

dedicated phosphor imaging plates. The regions of interest

255

(ROIs) were placed on the whole aorta autoradiographic images

256

to obtain total quantum level units (QL) and surface (mm

2

) in

257

each ROI. Autoradiographic signal intensity (QL/mm

2

) was

258

measured in the whole aortas and compared between the

259

different groups. Following autoradiography, samples were

260

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261

frozen at − 20 °C and then stained with Oil Red O (Sigma-

262

Aldrich) to visualize fat cells and neutral lipids.

263

Statistical analysis

264

All data are represented as mean ± standard error (SD) except

265

for autoradiographic quantification in which median and

266

interquartile range were used. Statistical significance between

267

experimental groups (P b 0.05) was assessed using GraphPad

268

Prism software (GraphPad Software, Inc., La Jolla, U.S.): P b

269

0.05 (*); P b 0.01 (**); P b 0.001 (***). DLS measurements

270

were compared using the Holm-Sidak method, without assuming

271

a consistent SD. Biodistribution data were compared using the

272

two-stage linear step-up procedure of Benjamini, Krieger and

273

Yekutieli, with Q = 1%. Each organ was analyzed individually,

274

without assuming a consistent SD. A two-tailed Mann-Whitney

275

test was used to compare uptake of NLC in whole aortas among

276

the different groups.

277

Results

278

Particle synthesis and characterization

279

The NLC platform is composed of a lipid core, mixture of

280

mono-, di-, and tri-glycerides (C10-C18) (soybean oil/Suppocire ™

281

NB), surrounded by a surfactant shell composed of phospholipids

282

(soybean lecithin s75) and PEGylated surfactants (Myrj ™ s40)

283

(Figure 1, A). A design of experiments optimized the component

284

ratios to yield very stable colloidal formulations.

24,33

To confer the

285

NLC platform with radioelement chelating properties to follow its

286

biodistribution and atherosclerotic plaque accumulation in mice

287

models, B22280, an amphiphilic DOTA moiety, was synthetized

288

and introduced in the nanoparticle formulation (Figure 1, A).

B22280-NLC were produced by ultrasonication and purified by

289

dialysis (Figure 1, B) as previously described for similar “nude” or

290

drug-loaded nanoparticles.

24,33

The ultrasonication/dialysis pro-

291

cess yielded very reproducible formulations of round-shaped

292

nanoparticles of mean hydrodynamic diameter of 51.5 ± 4.5 nm

293

and mean dispersity index of 0.165 ± 0.021 (Figure 2, A, B, C).

294

Though this process is very satisfactory for NLC lab scale

295

production, it is not translatable into an industrial larger scale

296

production process. Therefore, high pressure homogenization

297

(HPH) followed by tangential flow filtration (TFF) purification

298

was developed as an alternative production process, compatible

299

with mid to large scale production (50 g of particles were treated by

300

HPH, 15 g by TFF) (Figure 1, C). After process optimization, NLC

301

could be obtained in about 50 min (16 HPH cycles) (Figure 1, C).

302

After purification by TFF, HPH-produced B22280-NLC displayed

303

similar shape, hydrodynamic diameter, and zeta potential as

304

nanoparticles obtained by ultrasonication (Figure 2). Remarkably,

305

HPH dispersions presented improved monodispersity (dispersity

306

index of 0.066 ± 0.016 versus 0.165 ± 0.021 for ultrasonication)

307

and batch reproducibility (Figure 2).

308

Long-term stability of B22280-NLC upon storage at 4 °C,

309

100 mg/mL, in “ready-to-chelate” acetate buffer pH 8.6, was

310

assessed by DLS analysis. Nanoparticle dispersions obtained by

311

both processes were stable for more than 1 year in these storage

312

conditions (Figure 2, B and E, Figures S3-S6 in supplementary

313

materials). Endotoxin levels were below 1 EU/mL (for particle

314

concentration at 100 mg/mL) for 0.22-μm-filtered particle

315

dispersions.

316

Particle labeling

317

64

Cu chelating experiments were performed by incubating

318

nanoparticles at 60 °C for 25 min in presence of

64

CuCl

2

in

319

ammonium acetate buffer followed by 0.22 μm sterile filtration.

320

Figure 1. Structure of B22280-NLC (A) and their formulation process by ultrasonication/dialysis at lab-scale (B), or by HPH/TFF at larger scale (C). Particle size

decrease during process (ultrasonication, HPH) is evidenced by naked eye (milky to translucent transition) and DLS measurement.

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321 64

Cu was efficiently and specifically chelated by B22280-NLC, as

322

attested by the whole amount of radioactivity collected in the first

323

fractions in size exclusion chromatography experiments, contrary

324

to “naked” nanoparticles and

64

Cu-EDTA. (Figure 3, A). Post-

325

filtration radiochemical purity of

64

Cu-B22280-NLC was over

326

99% (Figure 3, B, C) and the filter remaining activity was always

327

inferior to 5%. The radiolabeling procedure did not change the

328

hydrodynamic diameter (46.3 ± 2.0 nm for radiolabeled B22280-

329

NLC, vs 49.2 ± 0.3 nm for B22280-NLC before labeling, P N 0.05)

330

or the dispersity index (0.128 ± 0.010 for radiolabeled B22280-

331

NLC vs 0.133 ± 0.005 for B22280-NLC before labeling, P N 0.05)

332

as assessed by DLS.

333

In vivo particle biodistribution

334

On PET/MRI acquisition performed 4 h and 24 h p.i. of

64

Cu-

335

B22280-NLC (n = 4), nanoparticles accumulated predominantly

336

in the liver (15.6 ± 1.5 and 21.4 ± 1.3 %ID/g), lungs (26.0 ± 3.5

337

and 10.8 ± 2.0 %ID/g), kidneys (6.0 ± 0.8 and 4.4 ± 0.8 %ID/g),

338

spleen (10.4 ± 1.1 and 9.7 ± 1.8 %ID/g), blood (14.3 ± 2.2 and

339

4.6 ± 2.0 %ID/g) and heart (8.2 ± 1.7 and 3.7 ± 0.9 %ID/g)

340

(Figure 4). The intensity of the signal measured in organs varied

341

significantly between 4 h and 24 h p.i. in lungs (P b 0.001), liver,

342

blood, heart and muscle (P b 0.01). The increase in liver uptake

343

together with a decrease in the other organs was compatible with

344

a predominant hepatobiliary clearance.

345

PET/MR imaging

346

Focal signals were detected in the aortic region on PET

347

images acquired 4 h p.i. of

64

Cu-B22280-NLC from ApoE

−/−

348

mice (Figure S7 in supplementary materials) that decreased on

349

the acquisitions performed 24 h p.i. Although liver uptake

350

hampered aortic root and arch uptake analysis, the focal signals

351

were predominantly observed in the aortic bifurcation and

352

carotid arteries, regions known as typical atherosclerotic lesions

353

localizations in this animal model.

34

Autoradiography, histology

354

Autoradiography allowed clear visualization of

64

Cu-

355

B22280-NLC uptake in the ApoE

−/−

mice aortas compared to

356

controls (Figure 5). Moreover, there was a strong co-localization

357

between radioactive signal and Oil Red O staining positive areas

358

(Figure 5, C, D, E, F). Quantification of autoradiographic

359

images comparing signal intensity in the whole aortas from the

360

different groups is presented in Figure 5, G. Autoradiographic

361

signal intensity of

64

Cu-B22280-NLC was significantly greater

362

in the ApoE

−/−

group (8.95 [7.58, 10.16] × 10

8

QL/mm

2

)

363

compared to the ApoE

−/−

control group (1.77 [1.46,

364

1.83] × 10

8

QL/mm

2

, P b 0.01) and the WT control group

365

(4.59 [3.11, 5.03] × 10

8

QL/mm

2

, P b 0.05).

366

Nanoparticle biocompatibility and tolerance (in vitro and in

367

vivo results)

368

Terminal complement complex SC5b-9 formation in human

369

sera upon incubation with nanoparticles in vitro has been used as

370

a predictor of complement activation-related hypersensitivity

371

reactions to nanoparticles. Therefore NLC and B22280-NLC

372

were tested at low and high concentrations (25 and 250 μ g/mL)

373

for complement activation in five human sera (in vitro).

374

Measured SC5b-9 concentrations are reported in Table 1 in

375

comparison to PBS and zymosan used as negative and positive

376

activation control, respectively. No complement activation by

377

any of the samples was observed.

378

Additional biocompatibility experiments were performed to

379

assess potential complement activation-related pseudoallergy

380

(CARPA) in 3 pigs. Just as in humans, the symptoms of HSR in

381

pigs arise within minutes after intravenous administration of

382

reactive nanoparticles and the reactions subside within 15-60

383

min. The most prominent CARPA symptom is a transient

384

elevation in pulmonary arterial pressure (PAP), rising up to 3%-

385

400% within 2-3 min after bolus injection and usually returning

386

to baseline within 10-20 min. Another marker is the change in

387

Figure 2. Characterization of B22280-NLC obtained by ultrasonication/dialysis (A, B, C) or HPH/TFF (D, E, F): TEM images (A, D); DLS profile examples (B,

E) and data summary (C, F).

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UNCORRECTED PR

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the plasma level of thromboxane B2 (TXB2), the stable

388

metabolite of TXA2. Rises in TXB2 and PAP usually closely

389

correlate, the latter being the consequence of the vasoconstrictor

390

TXA2 release.

29

CARPA experiments were carried out with

391

B22280-NLC particles chelated or not with non-radioactive Cu

2 392 +

. Based on our previous experience with liposomes or NLC,

30 393

slow infusion was selected here as way of administration (using a

394

rate of 2.5 mL/min). Figure 6, A and B shows the hemodynamic

395

and TXB2 effects of infusion with the chelated Cu-B22280-

396

NLC. Saline was infused (for 5 min) at the beginning of the

397

experiment as negative control, whereas zymosan (0.1 mg/kg)

398

was injected at the end of experiments as complement activating

399

positive control. In Figure 6, A there was an apparent increase

400

(up to 50%) in PAP during the infusion of low and high (5-fold)

401

dose of NLC. TXB2 levels did not change, which suggest that

402

the minor pulmonary hypertension was not mediated by TXB2.

403

Figure 6, B shows a similar experiment, but here only 2-fold

404

dose represented high dose. Only marginal changes in both PAP

405

Figure 3. Characterization of

64

Cu radiolabeled B22280-NLC. (A) Size exclusion chromatography profiles expressed in percentage of total activity after incubation with

64

Cu of B22280-NLC (blue) (chelation of

64

Cu on the surface of particles, eluted in the first fractions), NLC (red) (absence of chelation of

64

Cu on the particle surface) and EDTA (green). Thin layer chromatography of

64

Cu-B22280-NLC (Rf ≈ 0) (B), compared to free

64

Cu (Rf ≈ 1) as a control (C).

Figure 4. In vivo biodistribution of

64

Cu-B22280-NLC based on PET/MR

images analysis. Biodistribution data (mean and SD) are indicated in

percentage of injected dose per gram of organs (%ID/g) in ApoE

−/−

mice (n =

4) at 4 h and 24 h p.i.

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UNCORRECTED PR

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Figure 5. Autoradiography and Oil Red O staining of mice aortas. (A) Aorta from control WT mouse receiving

64

Cu-B22280-NLC. (B) Aorta from ApoE

−/−

mouse receiving control

64

Cu. (C) Aorta from ApoE

−/−

mouse receiving

64

Cu-B22280-NLC. (D) Oil red O staining of the same aorta represented in C. Blue arrows in C and D show the co-localization between the signal detected with autoradiography and the presence of lipids as attested by Oil red O staining. (E and F) Oil Red O staining and autoradiography of the same slide from the aortic root of a ApoE

−/−

mice injected with

64

Cu-B22280-NLC. Yellow stars indicate the correspondence between the presence of lipid deposits (E) and the uptake of

64

Cu-B22280-NLC (F). (G) Comparison of signals quantification from autoradiographic data expressed as intensity per unit area of the whole aorta (whole aorta autoradiographic signal/whole aorta area, QL/mm

2

).

Table 1 t1:1

Complement activation tests in human sera as assessed by SC5b-9 concentrations (μg/mL).

t1:2

t1:3 PBS Zymosan NLC

250 μg/mL

NLC 25 μg/mL

B22280-NLC 250 μg/mL

B22280-NLC 25 μg/mL

t1:4 #1 3.08 106.08 3.85 3.64 3.38 3.18

t1:5 #2 2.44 107.15 3.63 3.18 3.05 2.71

t1:6 #3 2.88 126.32 2.49 2.78 2.04 2.35

t1:7 #4 1.13 77.17 1.08 0.97 1.08 0.99

t1:8 #5 3.74 152.79 3.02 4.09 3.12 2.21

t1:9 Average 2.652 113.901 2.813 2.934 2.534 2.286

t1:10 Standard deviation 0.97 27.94 1.11 1.20 0.96 0.82

t1:11 RSD (%) 36.6 24.5 39.4 40.9 37.9 35.7

NLC and B22280-NLC were incubated with human sera for 45 min at 37 °C and SC5b-9 was measured as described in the Methods.

t1:12

(10)

UNCORRECTED PR

OOF

(11)

UNCORRECTED PR

OOF

406

and TXB2 were seen, whereas zymosan elicited similarly strong

407

reaction as before. In Figure 6, C the effects of B22280-NLC are

408

depicted. Infusions with the low and high (5-fold) doses caused

409

only marginal changes in both PAP and TXB2, and even an i.v.

410

bolus injection (7.5 mg/kg) elicited small elevations in both PAP

411

and TXB2 values. In Figure 6, C zymosan again elicited strong

412

CARPA in both parameters under investigation.

413

Discussion

414

There is a dire clinical need to identify and specifically target

415

inflamed and presumably rupture-prone atherosclerotic plaques.

416

Functional and molecular imaging might contribute to select

417

high risk patients that should be referred for direct therapy to

418

prevent cardiovascular events.

35,36

The development and

419

validation of targeted drug delivery approaches by non-

420

invasive molecular imaging techniques also constitute a crucial

421

step in the booming field of athero-nanomedicine.

37,38

The

422

recent development of positron emitter such as Copper-64

39

and

423

Zirconium-89

40

exhibiting half-lives suitable with the assess-

424

ment of nanoparticle biodistribution allowed to overcome the

425

traditional limitation of PET isotopes displaying rapid radioac-

tive decay. Here we report on the development and validation of

426

a novel, flexible nanoparticle platform for atherosclerotic plaque

427

targeting demonstrated by PET imaging in combination with

428

MRI for spatial resolution and soft tissue contrast. PET/MRI

429

acquisitions 4 h p.i. showed uptake of

64

Cu-B22280-NLC in the

430

preferential localizations of plaque development in ApoE

−/− 431

mice such as carotid arteries and aorta.

34,37

In vivo visualization

432

of NLC uptake in the thoracic aorta was hampered by signal

433

coming from liver and lungs. However, autoradiography on

434

whole aortas 24 h p.i. revealed a significant uptake that co-

435

localized with lipid staining. The in vivo demonstration of NLC

436

uptake in a preclinical model of atherosclerosis opens the way to

437

further studies dedicated to the optimization of this platform,

438

particularly towards therapeutic purposes, because of the long

439

blood-circulation time of the particles.

440

The underlying biological processes leading to plaque

441

accumulation of NLC are still to be elucidated. Macrophage

442

phagocytic activity and lipoprotein metabolism are potentially

443

implicated biological pathways to investigate.

41

NLC present

444

strong structural similarity with chylomicrons in terms of lipid

445

composition, and with VLDL in terms of particle size. Since

446Q4

now, other lipoprotein classes, LDL and HDL, have attracted

447

attention for the design of imaging and drug delivery

448

Figure 6. Hemodynamic effects of Cu-B22280-NLC nanoparticles in pigs. Monitoring of systolic arterial pressure (SAP), pulmonary arterial pressure (PAP),

heart rate (HR) and plasma levels of thromboxane B2 (TXB2) following Cu-B22280-NLC (A and B: 5 and 2 fold dose increase, respectively) and B22280-NLC

(C) injection compared to saline and zymosan as negative and positive controls, respectively.

(12)

UNCORRECTED PR

OOF

449

nanoplatforms in the early detection/treatment of atherosclerotic

450

lesions and their stable/vulnerable classification. In early

451

atherosclerotic plaque formation, LDL particles enter the

452

dysfunctional endothelium and accumulate in the intima of the

453

arterial wall, a feature that can be used to design efficient

454

targeting contrast agents.

42

HDL are reverse-cholesterol trans-

455

porters from peripheral tissues, including lipid-loaded macro-

456

phages, to the liver, and as such present athero-protective

457

properties.

43–46

They have been engineered to encapsulate

458

different drugs

18,47,48

and contrast agents.

49

Surprisingly, very

459

few studies have explored the potential of solid lipid nanopar-

460

ticles or nanostructured lipid carriers for atherosclerotic plaque

461

targeting since now, despite structural and composition similar-

462

ities with HDL and LDL. Solid lipid nanoparticles loaded with

463

iron oxide particles and PG12 prostacyclin, and stabilized with

464

nucleolipids, were demonstrated to reduce in vitro platelet

465

aggregation.

50

Lipid nanoparticles prepared by microfluidization

466

and loaded with paclitaxel-oleate

41

or carmustine

51

were shown

467

to reduce atherosclerosis lesions in rabbits. Wickline’s group has

468

also explored the potential of slightly different nanoparticles,

469

based on a perfluorocarbon oily core, for MR atherosclerosis

470

imaging

52

but also for therapeutic purposes.

53

Gu et al prepared

471

by a nanoprecipitation/solvent diffusion method lovastatin-

472

loaded NLC (LT-NLC) whose core was composed of cholesteryl

473

oleate, glycerol trioleate, lecithin, cholesterol and drug.

54

474

Interestingly, they evaluated in vitro the effect of coating the

475

nanoparticle shell with native HDL by incubating the particle

476

dispersion in the presence of the protein extracted from donor

477

blood. The authors evaluated the ability of LT-NLC and LT-

478

NLC-HDL to target raw macrophages and macrophage-derived

479

foam cells with/without enhancement of the VLDL receptor

480

pathway.

54

While LT-NLC were more internalized than LT-

481

NLC-HDL in raw macrophages, LT-NLC-HDL delivered higher

482

drug payload than LT-NLC in foam cells, and the process was

483

enhanced in the presence of lipase and VLDL. The authors

484

concluded that possible remodeling of LT-NLC-HDL could

485

occur in the presence of VLDL and lipase, resulting in partial

486

drug transfer into VLDL able to efficiently target foam cells

487

through VLDL receptors expressed at their surface.

54

Similarly,

488

B22280-NLC could interact with blood circulating lipoproteins

489

just after injection, resulting in surface remodeling with

490

apolipoprotein coating and/or partial transfer of contrast payload

491

to lipoproteins. As such, NLC could be particles of choice to act

492

as lipoprotein mimics targeting atherosclerotic lesions, without

493

drawbacks of native, recombinant or engineered lipoproteins, i.e.

494

complex, poorly controlled and expensive production or

495

isolation process.

496

Indeed, the HPH process used herein for NLC production is

497

water-based (i.e. solvent-free) and easy-to-implement.

55–57

498

Batches of various volumes (from ten of milliliters to liters)

499

can be produced without major protocol adaptation using

500

different homogenizers fit to the batch size.

26

If the high

501

relevance and interest of nanomedicines for the detection and

502

treatment of cardiovascular diseases were underlined in recent

503

reviews published by different groups,

7–11

nanoparticle large-

504

scale production with Good Manufacturing Practice (GMP),

505

batch characterization and qualification according to criteria still

506

to be clearly defined, and potential nanosystem toxicity, are still

challenging hurdles to overcome for clinical translation.

11,58 507

Batch production and characterization challenges could be easily

508

mastered for NLC production, as demonstrated here. Interest-

509

ingly, particle dispersion features (particle diameter, dispersity

510

index and storage lifetime) were improved by batch scaling using

511

HPH process.

26 512

NLC also present interesting features with regards to potential

513

toxicity and adverse effects following administration.

59,60

They

514

mainly employ ingredients of natural origin such as oils, fatty

515

acids, triglycerides, waxes, phospholipids, well-known for their

516

high biocompatibility and tolerability for medical applications.

517

Potential adverse effects of intravenous administration of NLC

518

were explored by complement activation in human sera in vitro

519

and CARPA experiments in pigs. In the latter, NLC were

520

administered as slow infusion, since previous unpublished data

521

showed that NLC (and liposomes as well) could induce CARPA

522

if injected as bolus. Under these conditions NLC caused no, or

523

minor hemodynamic changes, which, together with the lack of

524

complement activation in human serum in vitro imply no, or

525

small risk of infusion reactions in vivo (if infused slowly). It is

526

known that the porcine CARPA model is very sensitive for

527

cardiopulmonary distress, mimicking the sensitivity of hyper-

528

sensitive humans.

29

Therefore, these experiments, when suitably

529

translated, are giving a warning to clinicians (in accordance with

530

EMA’s requirements) that an occasional infusion reaction cannot

531

be entirely ruled out. This implies that the drug should be slowly

532

infused with close attention to any sign of infusion reaction.

533

Alternatively, an in vitro test of major complement activation by

534

the drug in the patient’s serum could serve as a predictor of

535

individual risk for CARPA.

536

In conclusion, B22280-NLC were demonstrated to accumu-

537

late in atherosclerotic lesions and could constitute interesting

538

nanosystems to explore as mimics and alternatives to native or

539

recombined lipoproteins. Their main advantage resides in a

540

better-mastered and up-scalable production process, in the

541

perspective of market production and clinical translation.

542

However, further experiments should be performed in order to

543

confirm their potential as drug or contrast agent carriers in the

544

context of cardiovascular diseases, in particular concerning their

545

safety profile.

546

Appendix A. Supplementary data

547

Supplementary data to this article can be found online at

548

https://doi.org/10.1016/j.nano.2020.102157.

549

References

550

1. Herrington W, Lacey B, Sherliker P, Armitage J, Lewington S.

551

Epidemiology of atherosclerosis and the potential to reduce the global

552

burden of atherothrombotic disease. Circ Res 2016;118:535-546.

553

2. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease.

554

N Engl J Med 2005;352:1685-1695.

555

3. Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W, Rosenfeld ME,

556

et al. A definition of initial, fatty streak, and intermediate lesions of

557

atherosclerosis. A report from the Committee on Vascular Lesions of the

558

Council on Arteriosclerosis, American Heart Association. Circulation

559

1994;89:2462-2478.

560

(13)

UNCORRECTED PR

OOF

561

4. Muller JE, Abela GS, Nesto RW, Tofler GH. Triggers, acute risk factors

562

and vulnerable plaques: the lexicon of a new frontier. J Am Coll Cardiol

563

1994;23:809-813.

564

5. Chono S, Tauchi Y, Deguchi Y, Morimoto K. Efficient drug delivery to

565

atherosclerotic lesions and the antiatherosclerotic effect by dexameth-

566

asone incorporated into liposomes in atherogenic mice. J Drug Target

567

2005;13:267-276.

568

6. van der Valk FM, van Wijk DF, Lobatto ME, Verberne HJ, Storm G,

569

Willems MCM, et al. Prednisolone-containing liposomes accumulate in

570

human atherosclerotic macrophages upon intravenous administration.

571

Nanomed Nanotechnol Biol Med 2015;11:1039-1046.

572

7. Nguyen LTH, Muktabar A, Tang J, Dravid VP, Thaxton CS, Venkatraman

573

S, et al. Engineered nanoparticles for the detection, treatment and

574

prevention of atherosclerosis: how close are we? Drug Discov Today

575

2017;22:1438-1446.

576

8. Nakhlband A, Eskandani M, Omidi Y, Saeedi N, Ghaffari S, Barar J, et

577

al. Combating atherosclerosis with targeted nanomedicines: recent

578

advances and future prospective. BioImpacts BI 2018;8:59-75.

579

9. Cicha I, Chauvierre C, Texier I, Cabella C, Metselaar JM, Szebeni J, et

580

al. From design to the clinic: practical guidelines for translating

581

cardiovascular nanomedicine. Cardiovasc Res 2018;114:1714-1727.

582

10. Schiener M, Hossann M, Viola JR, Ortega-Gomez A, Weber C, Lauber

583

K, et al. Nanomedicine-based strategies for treatment of atherosclerosis.

584

Trends Mol Med 2014;20:271-281.

585

11. Mulder WJM, JafferFA, Fayad ZA, Nahrendorf M. Imaging and nanomedicine

586

in inflammatory atherosclerosis. Sci Transl Med 2014;6:239sr1.

587

12. Ventola CL. Progress in nanomedicine: approved and investigational

588

nanodrugs. Pharm Ther 2017;42:742-755.

589

13. Chung EJ, Tirrell M. Recent advances in targeted, self-assembling

590

nanoparticles to address vascular damage due to atherosclerosis. Adv

591

Healthcare Mater 2015;4:2408-2422.

592

14. Skajaa T, Cormode DP, Falk E, Mulder WJM, Fisher EA, Fayad ZA.

593

High density lipoprotein-based contrast agents for multimodal imaging

594

of atherosclerosis. Arterioscler Thromb Vasc Biol 2010;30:169-176.

595

15. Shaish A, Keren G, Chouraqui P, Levkovitz H, Harats D. Imaging of

596

aortic atherosclerotic lesions by (125)I-LDL, (125)I-oxidized-LDL,

597

(125)I-HDL and (125)I-BSA. Pathobiol J Immunopathol Mol Cell

598

Biol 2001;69:225-229.

599

16. Almer G, Mangge H, Zimmer A, Prassl R. Lipoprotein-related and

600

apolipoprotein-mediated delivery systems for drug targeting and

601

imaging. Curr Med Chem 2015;22:3631-3651.

602

17. Tauchi Y, Zushida L, Chono S, Sato J, Ito K, Morimoto K. Effect of

603

dexamethasone palmitate-low density lipoprotein complex on choles-

604

terol ester accumulation in aorta of atherogenic model mice. Biol Pharm

605

Bull 2001;24:925-929.

606

18. Duivenvoorden R, Tang J, Cormode DP, Mieszawska AJ, Izquierdo-

607

Garcia D, Ozcan C, et al. A statin-loaded reconstituted high-density

608

lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation.

609

Nat Commun 2014;53065.

610

19. Leeper NJ, Park S-M, Smith BR. High-density lipoprotein nanoparticle

611

imaging in atherosclerotic vascular disease. JACC Basic Transl Sci

612

2017;2:98-100.

613

20. Teixeira MC, Carbone C, Souto EB. Beyond liposomes: recent advances

614

on lipid based nanostructures for poorly soluble/poorly permeable drug

615

delivery. Prog Lipid Res 2017;68:1-11.

616

21. Müller RH, Shegokar R, Keck CM. 20 years of lipid nanoparticles (SLN

617

and NLC): present state of development and industrial applications.

618

Curr Drug Discov Technol 2011;8:207-227.

619

22. Beloqui A, Solinís MÁ, Rodríguez-Gascón A, Almeida AJ, Préat V.

620

Nanostructured lipid carriers: promising drug delivery systems for future

621

clinics. Nanomedicine 2016;12:143-161.

622

23. Mérian J, Boisgard R, Decleves X, Thezé B, Texier I, Tavitian B.

623

Synthetic lipid nanoparticles targeting steroid organs. J Nucl Med

624

2013;54:1996-2003.

625

24. Delmas T, Couffin A-C, Bayle PA, de Crécy F, Neumann E, Vinet F, et

626

al. Preparation and characterization of highly stable lipid nanoparticles

with amorphous core of tuneable viscosity. J Colloid Interface Sci

627

2011;360:471-481.

628

25. Göppert TM, Müller RH. Adsorption kinetics of plasma proteins on solid

629

lipid nanoparticles for drug targeting. Int J Pharm 2005;302:172-186.

630

26. Jenning V, Lippacher A, Gohla SH. Medium scale production of solid

631

lipid nanoparticles (SLN) by high pressure homogenization. J Micro-

632

encapsul 2002;19:1-10.

633

27. Data requirements for intravenous liposomal products developed with

634

reference to an innovator product [Internet]Eur Med Agency 2018 [cited

635

2019 Apr 22]. Available from:, https://www.ema.europa.eu/en/data-

636

requirements-intravenous-liposomal-products-developed-reference-

637

innovator-liposomal-product .

638Q6

28. Szebeni J. Complement activation-related pseudoallergy: a stress

639

reaction in blood triggered by nanomedicines and biologicals. Mol

640

Immunol 2014;61:163-173.

641

29. Szebeni J, Urbanics R, Dézsi L, Bedőcs P. A porcine model of

642

complement activation-related pseudoallergy to nano-pharmaceuticals:

643

pros and cons of translation to a preclinical safety test. Precis

644

Nanomedicine 2018;1:63-73.

645

30. Szebeni J, Bedőcs P, Csukás D, Rosivall L, Bünger R, Urbanics R. A

646

porcine model of complement-mediated infusion reactions to drug

647

carrier nanosystems and other medicines. Adv Drug Deliv Rev

648

2012;64:1706-1716.

649

31. Gravier J, Navarro FP, Delmas T, Mittler F, Couffin A-C, Vinet F, et al.

650

Lipidots: competitive organic alternative to quantum dots for in vivo

651

fluorescence imaging. J Biomed Opt 2011;16096013.

652

32. Caputo F, Arnould A, Bacia M, Ling WL, Rustique E, Texier I, et al.

653

Measuring particle size distribution by asymmetric flow field flow

654

fractionation: a powerful method for the preclinical characterization of

655

lipid-based nanoparticles. Mol Pharm 2019;16:756-767.

656

33. Delmas T, Fraichard A, Bayle P-A, Texier I, Bardet M, Baudry J, et al.

657

Encapsulation and release behavior from lipid nanoparticles: model

658

study with Nile red fluorophore. J Colloid Sci Biotechnol 2012;1:16-25.

659

34. Meir Karen S, Leitersdorf Eran. Atherosclerosis in the apolipoprotein E-

660

deficient mouse. Arterioscler Thromb Vasc Biol 2004;24:1006-1014.

661

35. Wildgruber M, Swirski FK, Zernecke A. Molecular imaging of

662

inflammation in atherosclerosis. Theranostics 2013;3:865-884.

663

36. Jaffer FA, Libby P, Weissleder R. Molecular and cellular imaging of

664

atherosclerosis.emerging applications J Am Coll Cardiol2006;47:1328-1338.

665

37. Vigne J, Thackeray J, Essers J, Makowski M, Varasteh Z, Curaj A, et al.

666

Current and emerging preclinical approaches for imaging-based

667

characterization of atherosclerosis. Mol Imaging Biol 2018;20:869-887.

668

38. Chan CKW, Zhang L, Cheng CK, Yang H, Huang Y, Tian XY, et al.

669

Recent advances in managing atherosclerosis via nanomedicine. Small

670

Weinh Bergstr Ger 2018:14.

671

39. Nahrendorf M, Zhang H, Hembrador S, Panizzi P, Sosnovik DE, Aikawa

672

E, et al. Nanoparticle PET-CT imaging of macrophages in inflammatory

673

atherosclerosis. Circulation 2008;117:379-387.

674

40. Majmudar MD, Yoo J, Keliher EJ, Truelove JJ, Iwamoto Y, Sena B, et

675

al. Polymeric nanoparticle PET/MR imaging allows macrophage

676

detection in atherosclerotic plaques. Circ Res 2013;112:755-761.

677

41. Freitas SCMP, Tavares ER, Silva BMO, Meneghini BC, Kalil-Filho R,

678

Maranhão RC. Lipid core nanoparticles resembling low-density

679

lipoprotein and regression of atherosclerotic lesions: effects of particle

680

size. Braz J Med Biol Res 2018;51:1-8.

681

42. Frias JC, Lipinski MJ, Lipinski SE, Albelda MT. Modified lipoproteins

682

as contrast agents for imaging of atherosclerosis. Contrast Media Mol

683

Imaging 2007;2:16-23.

684

43. Zheng KH, Stroes ESG. HDL infusion for the management of

685

atherosclerosis: current developments and new directions. Curr Opin

686

Lipidol 2016;27:592-596.

687

44. Kuai R, Li D, ChenYE, Moon JJ, Schwendeman A. High-density lipoproteins:

688

nature’s multifunctional nanoparticles. ACS Nano 2016;10:3015-3041.

689

45. Cao Y-N, Xu L, Han Y-C, Wang Y-N, Liu G, Qi R. Recombinant high-

690

density lipoproteins and their use in cardiovascular diseases. Drug

691

Discov Today 2017;22:180-185.

692

(14)

UNCORRECTED PR

OOF

693

46. Guo Y, Yuan W, Yu B, Kuai R, Hu W, Morin EE, et al. Synthetic

694

high-density lipoprotein-mediated targeted delivery of liver X

695

receptors agonist promotes atherosclerosis regression. EBioMedicine

696

2018;28:225-233.

697

47. Wang K, Yu C, Liu Y, Zhang W, Sun Y, Chen Y. Enhanced

698

antiatherosclerotic efficacy of statin-loaded reconstituted high-density

699

lipoprotein via ganglioside GM1 modification. ACS Biomater Sci Eng

700

2018;4:952-962.

701

48. Jiang C, Zhao Y, Yang Y, He J, Zhang W, Liu J. Evaluation of the

702

combined effect of recombinant high-density lipoprotein carrier and the

703

encapsulated lovastatin in RAW264.7 macrophage cells based on the

704

median-effect principle. Mol Pharm 2018;15:1017-1027.

705

49. Cormode DP, Jarzyna PA, Mulder WJM, Fayad ZA. Modified natural

706

nanoparticles as contrast agents for medical imaging. Adv Drug Deliv

707

Rev 2010;62:329-338.

708

50. Oumzil K, Ramin MA, Lorenzato C, Hémadou A, Laroche J, Jacobin-

709

Valat MJ, et al. Solid lipid nanoparticles for image-guided therapy of

710

atherosclerosis. Bioconjug Chem 2016;27:569-575.

711

51. Daminelli EN, Martinelli AEM, Bulgarelli A, Freitas FR, Maranhão RC.

712

Reduction of atherosclerotic lesions by the chemotherapeutic agent

713

carmustine associated to lipid nanoparticles. Cardiovasc Drugs Ther

714

2016;30:433-443.

715

52. Palekar RU, Jallouk AP, Lanza GM, Pan H, Wickline SA. Molecular

716

imaging of atherosclerosis with nanoparticle-based fluorinated MRI

717

contrast agents. Nanomedicine 2015;10:1817-1832.

53. Palekar RU, Jallouk AP, Myerson JW, Pan H, Wickline SA. Inhibition of

718

thrombin with PPACK-nanoparticles restores disrupted endothelial

719

barriers and attenuates thrombotic risk in experimental atherosclerosis.

720

Arterioscler Thromb Vasc Biol 2016;36:446-455.

721

54. Gu X, Zhang W, Liu J, Shaw JP, Shen Y, Xu Y, et al. Preparation and

722

characterization of a lovastatin-loaded protein-free nanostructured lipid

723

carrier resembling high-density lipoprotein and evaluation of its

724

targeting to foam cells. AAPS PharmSciTech 2011;12:1200-1208.

725

55. Anton N, Benoit J-P, Saulnier P. Design and production of nanoparticles

726

formulated from nano-emulsion templates—a review. J Control Release

727

2008;128:185-199.

728

56. Zhang J, Fan Y, Smith E. Experimental design for the optimization of

729

lipid nanoparticles. J Pharm Sci 2009;98:1813-1819.

730

57. Severino P, Santana MHA, Souto EB. Optimizing SLN and NLC by 2(2)

731

full factorial design: effect of homogenization technique. Mater Sci Eng

732

C Mater Biol Appl 2012;32:1375-1379.

733

58. Cicha I, Lyer S, Alexiou C, Garlichs CD. Nanomedicine in diagnostics

734

and therapy of cardiovascular diseases: beyond atherosclerotic plaque

735

imaging. Nanotechnol Rev 2013;2:449-72.

736

59. Matuszak J, Baumgartner J, Zaloga J, Juenet M, da Silva AE, Franke D, et

737

al. Nanoparticles for intravascular applications: physicochemical charac-

738

terization and cytotoxicity testing. Nanomedicine 2016;11:597-616.

739

60. Matuszak J, Dörfler P, Lyer S, Unterweger H, Juenet M, Chauvierre C, et al.

740

Comparative analysis of nanosystems’ effects on human endothelial and

741

monocytic cell functions. Nanotoxicology 2018:1-18.

742

743 744

(15)

Q5

(16)

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