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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�
UNCORRECTED PR
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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
6ApoE
−/−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
UNCORRECTED PR
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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
64Cu-fonctionalized NLC in
20
atherosclerotic mice.
64Cu-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
64Cu-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
8vs 4.59 [3.11, 5.03] ×10
8QL/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
29diseases and still represents a major burden globally.
1Atheroscle-
30rotic lesions are mainly composed of cells (especially inflamma-
31tory and immune cells), connective tissue elements, debris and
32lipids within the arterial wall.
2Advanced lesions contain abnormal
33accumulation of lipoproteins and cholesterol esters (CE) with
34concomitant chronic inflammation.
3Atherosclerotic plaques
35prone to rupture, also called vulnerable plaques, are usually
36characterized by the presence of a lipid-rich necrotic core that is
37covered by a thin fibrous cap and depleted of smooth muscle cells.
4 38There is a need to develop strategies to manage plaque
39treatment more effectively and establish precise diagnosis on
40disease development status. Nanotechnology-based approaches
41for targeted drug delivery have now entered clinical practice in the
42field of oncology. Nanomedicine formulations have recently
43demonstrated their interest to deliver efficiently active compounds
44Nanomedicine: 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.
5–11Liposomes 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.
7,8,12,13Lipoprotein-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.
14–1653
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.
1756
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.
1859
Lipoproteins are intrinsically biocompatible, biodegradable,
60
poorly immunogenic, and enable natural targeting properties.
18,1961
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.
1668
Nanostructured lipid carriers (NLC) have aroused high interest as
69
drug delivery systems for the last 25 years.
20–22NLC were found to
70
accumulate within lipid-recruiting tissues.
23and 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.
16However, 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.
24This 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.
2684
Herein, full process and methodology for scaled-up produc-
85
tion and quality control of
64Cu-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,
27especially using the
93
sensitive porcine complement activation-related pseudoallergy
94
(CARPA) model.
28–3095
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
100soybean oil from Croda Uniqema (Chocques, France). B22280
101chelate was synthetized as described in supplementary materials.
102Copper-64 (t
1/2= 12.7 h, β
+; 17.8%, Eβ
+max = 656 keV, β
−,
10338.4%, Eβ
−max = 573 keV) was obtained from ARRONAX
104cyclotron facility (GIP ARRONAX, Saint-Herblain, France)
105using the reaction
64Ni (p,n)
64Cu and was delivered as
64CuCl
2 106in 0.1 M HCl. Product was filtered through sterile Acrodisc®
107syringe filters (0.2 μm, 13 mm) with Supor® membrane (PALL
108Corporation, USA) before use. Other chemicals were purchased
109from Sigma-Aldrich (Saint-Quentin-Fallavier, France).
110Lipid nanoparticle synthesis
111Lipid nanoparticles were prepared at the lab scale (750 mg of
112particles) by ultrasonication similarly to previously described
113protocols,
24,31as well as by high pressure homogenization
114(HPH) at larger scale (50 g of particles). For ultrasonication, the
115lipid phase comprised 255 mg of soybean oil, 85 mg of
116Suppocire ™ NB, 65 mg of lecithin, and 5 mg of amphiphilic
117chelate B22280, and the aqueous phase 345 mg of Myrj ™ S40
118and water (qsp 2 mL). For HPH, ingredient quantities were
119scaled up 75 folds. Mixture of lipid and aqueous phases was pre-
120emulsified using a mechanical disperser (Ultra-T25 Digital
121Turrax, IKA) operated at 15,000 rpm for 5 min. The pre-
122emulsion was processed with a High Pressure Homogenizer
123(Panda Plus 2000, GEA Niro Soavi, Italy) operated for 16 cycles
124with a total pressure of 1200 bars, the pressure of the second
125stage 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
127followed by tangential flow filtration (Labscale TFF system,
128Millipore) against 1 M acetate buffer pH 8.3 through a Pellicon
129XL Biomax ™ cassette (Merck) operated at a trans-membrane
130pressure of 14 psi at a flow rate of 2 mL/min. The nanoparticle
131dispersion was adjusted to a concentration of 100 mg/mL and
132filtered through a 0.22 μm Millipore membrane for sterilization
133before storage and use.
134Lipid nanoparticle characterizations
135The endotoxin level in particle batches was measured by
136Charles River (Ecully, France) using the chromogenic LAL test.
137Dynamic light scattering (DLS) was used to determine the
138particle hydrodynamic diameter and zeta potential (Zeta Sizer
139Nano ZS, Malvern Instrument, Orsay, France). Particle disper-
140sions were diluted to 2 mg/mL of lipids in 0.22 μm filtered 0.1×
141PBS and transferred in Zeta Sizer Nano cells (Malvern
142Instrument) before each measurement, performed in triplicate.
143Results (Z-average diameter, dispersity index, zeta potential)
144were expressed as mean and standard deviation of three
145independent measurements performed at 25 °C. TEM images
146of NLC were obtained after negative staining (2% uranyl acetate)
147similarly to previously published protocol.
32 148Radiolabeling procedures and analysis
149150 μL of B22280-NLC (100 mg/mL of lipids) was incubated
150with 200 ± 20 MBq of
64CuCl
2and ammonium acetate buffer (0.1
151M, pH 7.0) in a rubber sealed vial. The mixture was heated at 60 °C
152for 25 min then filtered through a 0.22 μm membrane (IC Acrodisc,
153UNCORRECTED PR
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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
64Cu
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.
28Briefly, 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
208Institute (APAFIS #9681, Paris, France). ApoE
−/−mice (in-
209house breeding) aged over 35 weeks (weight 27-33 g) fed with
210normal diet and wild type (WT) C57BL/6 mice (Charles River
211laboratories) were housed under a 12 h light/dark cycle with free
212access to water and normal chow ad libitum. Three experimental
213groups were constituted: 1/ ApoE
−/−group (n = 6) receiving
214 64Cu-B22280-NLC (ApoE
−/−group, experimental model of
215atherosclerosis); 2/ WT group (n = 3) receiving
64Cu-B22280-
216NLC (WT control group) and 3/ ApoE
−/−(n = 5) group receiving
217 64CuCl
2(ApoE
−/−control group).
218MicroPET/MRI & imaging data analysis
219All PET/MRI scans were acquired using a Mediso NanoScan
220PET/MRI (1 T Mediso Ltd., Budapest, Hungary). PET
221acquisitions were performed 4 h and 24 h after retro-orbital
222injection of the radiotracer (≈20 MBq ; 100-150 μL ; 1.5-2.25
223mg NLC), followed by MRI acquisition (described in supple-
224mentary materials). PET data were reconstructed using Tera-
225Tomo™ 3D software (Mediso Ltd., Budapest, Hungary), a 3D-
226OSEM Monte Carlo based algorithm with attenuation and scatter
227corrections (voxel size equal to 0.3×0.3×0.3 mm
3). Intensity
228value in the voxels was calibrated in kilo Becquerel per cubic
229centimeter (kBq cm
−3) corrected for decay and positron
230branching ratio of
64Cu.
231PET and MR images were automatically co-registered and
232analyzed using PMOD software (v3.7, PMOD Technologies
233Ltd). Volumes of interest (VOI
s) were defined for the lungs,
234bladder, right kidney, heart, muscle on the thigh, stomach and a
235section of intestine on the MR images using threshold and
236manual drawing, and for liver and spleen on the PET images
237using threshold and manual drawing; blood activity was assessed
238in the left ventricle using a 1.5 cm
3ellipsoid volume. Percentage
239of administered dose per gram of tissue (%ID/g) was calculated
240for each organ using following equation:
241%ID=g ¼ Mean activity in VOI kBq cm ð
−3Þ =Organ density g cm ð
−3Þ Injected activity kBq ð Þ
242 243244
The injected activity was corrected for decay and reference time was the PET acquisition.
245Autoradiography, histology
246Immediately after 24 h PET/MRI acquisition, mice were
247euthanized with an overdose of isoflurane. Whole aortas were
248collected from aortic root to bifurcation and washed with saline.
249Aortas were dissected by removing adventitial adipose tissue
250under binocular microscope and opened longitudinally to be lied
251on slides. Radioactive signal on whole aortas was quantified
252using a calibrated bio-image analyzer CR-35 Bio (Elysia-
253Raytest, Liège, Belgium) after exposing samples for 24 h on
254dedicated phosphor imaging plates. The regions of interest
255(ROIs) were placed on the whole aorta autoradiographic images
256to obtain total quantum level units (QL) and surface (mm
2) in
257each ROI. Autoradiographic signal intensity (QL/mm
2) was
258measured in the whole aortas and compared between the
259different groups. Following autoradiography, samples were
260UNCORRECTED PR
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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.
24,33To 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
289dialysis (Figure 1, B) as previously described for similar “nude” or
290drug-loaded nanoparticles.
24,33The ultrasonication/dialysis pro-
291cess yielded very reproducible formulations of round-shaped
292nanoparticles of mean hydrodynamic diameter of 51.5 ± 4.5 nm
293and mean dispersity index of 0.165 ± 0.021 (Figure 2, A, B, C).
294Though this process is very satisfactory for NLC lab scale
295production, it is not translatable into an industrial larger scale
296production process. Therefore, high pressure homogenization
297(HPH) followed by tangential flow filtration (TFF) purification
298was developed as an alternative production process, compatible
299with mid to large scale production (50 g of particles were treated by
300HPH, 15 g by TFF) (Figure 1, C). After process optimization, NLC
301could be obtained in about 50 min (16 HPH cycles) (Figure 1, C).
302After purification by TFF, HPH-produced B22280-NLC displayed
303similar shape, hydrodynamic diameter, and zeta potential as
304nanoparticles obtained by ultrasonication (Figure 2). Remarkably,
305HPH dispersions presented improved monodispersity (dispersity
306index of 0.066 ± 0.016 versus 0.165 ± 0.021 for ultrasonication)
307and batch reproducibility (Figure 2).
308Long-term stability of B22280-NLC upon storage at 4 °C,
309100 mg/mL, in “ready-to-chelate” acetate buffer pH 8.6, was
310assessed by DLS analysis. Nanoparticle dispersions obtained by
311both processes were stable for more than 1 year in these storage
312conditions (Figure 2, B and E, Figures S3-S6 in supplementary
313materials). Endotoxin levels were below 1 EU/mL (for particle
314concentration at 100 mg/mL) for 0.22-μm-filtered particle
315dispersions.
316Particle labeling
31764
Cu chelating experiments were performed by incubating
318nanoparticles at 60 °C for 25 min in presence of
64CuCl
2in
319ammonium acetate buffer followed by 0.22 μm sterile filtration.
320Figure 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.
UNCORRECTED PR
OOF
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
64Cu-EDTA. (Figure 3, A). Post-
325
filtration radiochemical purity of
64Cu-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
64Cu-
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
64Cu-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.
34Autoradiography, histology
354Autoradiography allowed clear visualization of
64Cu-
355B22280-NLC uptake in the ApoE
−/−mice aortas compared to
356controls (Figure 5). Moreover, there was a strong co-localization
357between radioactive signal and Oil Red O staining positive areas
358(Figure 5, C, D, E, F). Quantification of autoradiographic
359images comparing signal intensity in the whole aortas from the
360different groups is presented in Figure 5, G. Autoradiographic
361signal intensity of
64Cu-B22280-NLC was significantly greater
362in the ApoE
−/−group (8.95 [7.58, 10.16] × 10
8QL/mm
2)
363compared to the ApoE
−/−control group (1.77 [1.46,
3641.83] × 10
8QL/mm
2, P b 0.01) and the WT control group
365(4.59 [3.11, 5.03] × 10
8QL/mm
2, P b 0.05).
366Nanoparticle biocompatibility and tolerance (in vitro and in
367vivo results)
368Terminal complement complex SC5b-9 formation in human
369sera upon incubation with nanoparticles in vitro has been used as
370a predictor of complement activation-related hypersensitivity
371reactions to nanoparticles. Therefore NLC and B22280-NLC
372were tested at low and high concentrations (25 and 250 μ g/mL)
373for complement activation in five human sera (in vitro).
374Measured SC5b-9 concentrations are reported in Table 1 in
375comparison to PBS and zymosan used as negative and positive
376activation control, respectively. No complement activation by
377any of the samples was observed.
378Additional biocompatibility experiments were performed to
379assess potential complement activation-related pseudoallergy
380(CARPA) in 3 pigs. Just as in humans, the symptoms of HSR in
381pigs arise within minutes after intravenous administration of
382reactive nanoparticles and the reactions subside within 15-60
383min. The most prominent CARPA symptom is a transient
384elevation in pulmonary arterial pressure (PAP), rising up to 3%-
385400% within 2-3 min after bolus injection and usually returning
386to baseline within 10-20 min. Another marker is the change in
387Figure 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).
UNCORRECTED PR
OOF
the plasma level of thromboxane B2 (TXB2), the stable
388metabolite of TXA2. Rises in TXB2 and PAP usually closely
389correlate, the latter being the consequence of the vasoconstrictor
390TXA2 release.
29CARPA experiments were carried out with
391B22280-NLC particles chelated or not with non-radioactive Cu
2 392 +. Based on our previous experience with liposomes or NLC,
30 393slow infusion was selected here as way of administration (using a
394rate of 2.5 mL/min). Figure 6, A and B shows the hemodynamic
395and TXB2 effects of infusion with the chelated Cu-B22280-
396NLC. Saline was infused (for 5 min) at the beginning of the
397experiment as negative control, whereas zymosan (0.1 mg/kg)
398was injected at the end of experiments as complement activating
399positive 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)
401dose of NLC. TXB2 levels did not change, which suggest that
402the minor pulmonary hypertension was not mediated by TXB2.
403Figure 6, B shows a similar experiment, but here only 2-fold
404dose represented high dose. Only marginal changes in both PAP
405Figure 3. Characterization of
64Cu radiolabeled B22280-NLC. (A) Size exclusion chromatography profiles expressed in percentage of total activity after incubation with
64Cu of B22280-NLC (blue) (chelation of
64Cu on the surface of particles, eluted in the first fractions), NLC (red) (absence of chelation of
64Cu on the particle surface) and EDTA (green). Thin layer chromatography of
64Cu-B22280-NLC (Rf ≈ 0) (B), compared to free
64Cu (Rf ≈ 1) as a control (C).
Figure 4. In vivo biodistribution of
64Cu-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.
UNCORRECTED PR
OOF
Figure 5. Autoradiography and Oil Red O staining of mice aortas. (A) Aorta from control WT mouse receiving
64Cu-B22280-NLC. (B) Aorta from ApoE
−/−mouse receiving control
64Cu. (C) Aorta from ApoE
−/−mouse receiving
64Cu-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
64Cu-B22280-NLC. Yellow stars indicate the correspondence between the presence of lipid deposits (E) and the uptake of
64Cu-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
UNCORRECTED PR
OOF
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.
35,36The 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.
37,38The
422
recent development of positron emitter such as Copper-64
39and
423
Zirconium-89
40exhibiting 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
426a novel, flexible nanoparticle platform for atherosclerotic plaque
427targeting demonstrated by PET imaging in combination with
428MRI for spatial resolution and soft tissue contrast. PET/MRI
429acquisitions 4 h p.i. showed uptake of
64Cu-B22280-NLC in the
430preferential localizations of plaque development in ApoE
−/− 431mice such as carotid arteries and aorta.
34,37In vivo visualization
432of NLC uptake in the thoracic aorta was hampered by signal
433coming from liver and lungs. However, autoradiography on
434whole aortas 24 h p.i. revealed a significant uptake that co-
435localized with lipid staining. The in vivo demonstration of NLC
436uptake in a preclinical model of atherosclerosis opens the way to
437further studies dedicated to the optimization of this platform,
438particularly towards therapeutic purposes, because of the long
439blood-circulation time of the particles.
440The underlying biological processes leading to plaque
441accumulation of NLC are still to be elucidated. Macrophage
442phagocytic activity and lipoprotein metabolism are potentially
443implicated biological pathways to investigate.
41NLC present
444strong structural similarity with chylomicrons in terms of lipid
445composition, and with VLDL in terms of particle size. Since
446Q4now, other lipoprotein classes, LDL and HDL, have attracted
447attention for the design of imaging and drug delivery
448Figure 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.
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.
42HDL 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.
43–46They have been engineered to encapsulate
458
different drugs
18,47,48and contrast agents.
49Surprisingly, 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.
50Lipid nanoparticles prepared by microfluidization
466
and loaded with paclitaxel-oleate
41or carmustine
51were 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
52but also for therapeutic purposes.
53Gu 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.
54474
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.
54While 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.
54Similarly,
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.
55–57498
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.
26If 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,
7–11nanoparticle 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 507Batch production and characterization challenges could be easily
508mastered for NLC production, as demonstrated here. Interest-
509ingly, particle dispersion features (particle diameter, dispersity
510index and storage lifetime) were improved by batch scaling using
511HPH process.
26 512NLC also present interesting features with regards to potential
513toxicity and adverse effects following administration.
59,60They
514mainly employ ingredients of natural origin such as oils, fatty
515acids, triglycerides, waxes, phospholipids, well-known for their
516high biocompatibility and tolerability for medical applications.
517Potential adverse effects of intravenous administration of NLC
518were explored by complement activation in human sera in vitro
519and CARPA experiments in pigs. In the latter, NLC were
520administered as slow infusion, since previous unpublished data
521showed that NLC (and liposomes as well) could induce CARPA
522if injected as bolus. Under these conditions NLC caused no, or
523minor hemodynamic changes, which, together with the lack of
524complement activation in human serum in vitro imply no, or
525small risk of infusion reactions in vivo (if infused slowly). It is
526known that the porcine CARPA model is very sensitive for
527cardiopulmonary distress, mimicking the sensitivity of hyper-
528sensitive humans.
29Therefore, these experiments, when suitably
529translated, are giving a warning to clinicians (in accordance with
530EMA’s requirements) that an occasional infusion reaction cannot
531be entirely ruled out. This implies that the drug should be slowly
532infused with close attention to any sign of infusion reaction.
533Alternatively, an in vitro test of major complement activation by
534the drug in the patient’s serum could serve as a predictor of
535individual risk for CARPA.
536In conclusion, B22280-NLC were demonstrated to accumu-
537late in atherosclerotic lesions and could constitute interesting
538nanosystems to explore as mimics and alternatives to native or
539recombined lipoproteins. Their main advantage resides in a
540better-mastered and up-scalable production process, in the
541perspective of market production and clinical translation.
542However, further experiments should be performed in order to
543confirm their potential as drug or contrast agent carriers in the
544context of cardiovascular diseases, in particular concerning their
545safety profile.
546Appendix A. Supplementary data
547Supplementary data to this article can be found online at
548https://doi.org/10.1016/j.nano.2020.102157.
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