Squalenoyl adenosine nanoparticles provideneuroprotection after stroke and spinal cord injuryAlice Gaudin1,2, Müge Yemisci3, Hakan Eroglu4, Sinda Lepetre-Mouelhi1,2, Omer Faruk Turkoglu5,Buket Dönmez-Demir3, Seçil Caban4, Mustafa Fevzi Sargon6, Sébastien Garcia-Argote7,Grégory Pieters7, Olivier Loreau7, Bernard Rousseau7, Oya Tagit8, Niko Hildebrandt8,Yannick Le Dantec9, Julie Mougin1,2, Sabrina Valetti1,2, Hélène Chacun1,2, Valérie Nicolas10,Didier Desmaële1,2, Karine Andrieux1,2*, Yilmaz Capan4, Turgay Dalkara3 and Patrick Couvreur1,2*

There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such asstroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, areinefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream.Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allowsprolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. Theanimals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement oftheir neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case ofspinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extendadenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that ahydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a singleconjugation with the lipid squalene.

Brain diseases are a major health concern, due in part to anageing population and the life-long disability that theyimply1. Drug delivery remains the main challenge of central

nervous system (CNS) drug development2, because of the fast meta-bolization and/or rapid blood clearance of most CNS drugs, as wellas the generally poor diffusion through the blood–brain barrier(BBB) and blood–spinal cord barrier (BSCB). Another hurdle inCNS drug development is the high degree of complexity of thehuman brain3 and cerebral diseases4, which often involve complexprocesses featuring the interaction of multiple mechanismsbetween the cerebrovasculature and parenchyma5. It has beensuggested that nanotechnologies may promote the brain deliveryand efficacy of drugs6,7, as a result of improved pharmacokineticprofiles and better neurovascular unit access. However, many ofthese nanodevices require substantial design and complex multi-functionalization to achieve the targeted delivery of drugs, whichmay restrain their pharmaceutical development8. In addition, evenif a significant although limited BBB translocation has been demon-strated with these systems, there are only few examples of real sub-sequent pharmacological activity9. A typical example is adenosine, anucleoside with potentially significant beneficial activity in severalsevere neurological disorders10–13, but which has never been usedfor the treatment of cerebral diseases14 because of its short plasmahalf-life15, moderate side effects16–18 and its inability to cross theBBB and the BSCB19,20. Here, we report a very simple and easyway to use the currently unusable adenosine as a neuroprotective

drug following intravenous injection. We show that the bioconjuga-tion of adenosine with squalene (a natural and biocompatible lipid)to form an amphiphilic prodrug led to the spontaneous formationof nanoparticles with a size of ∼120 nm, allowing (1) efficientprotection from rapid metabolization, (2) induction of a dramaticneuroprotective effect in both an ischaemia–reperfusion model inmice and a spinal cord injury (SCI) model in rats, (3) a prolongeddrug interaction with the neurovascular unit, (4) no triggering ofside effects or systemic toxicity. This ‘squalenoylation’ technology,which has already been applied to the intravenous administrationof anticancer and antiretroviral compounds21–23, is shown here,for the first time, to be competent for the delivery of therapeuticamounts of drugs to treat CNS injuries.

Preparation and characterizationAs illustrated in Fig. 1a, the squalenylacetic acid has been covalentlylinked onto the amino group of the adenosine to form the prodrugsqualenoyl adenosine (Supplementary Section II.1). The nano-assemblies were prepared by nanoprecipitation of an ethanolic sol-ution of squalenoyl adenosine (SQAd) in a 5% aqueous dextrosesolution without any added surfactant, which from a toxicologicalpoint of view is a major advantage (Supplementary Fig. 1).Because adenosine is not physically encapsulated but is covalentlylinked to the lipid nanocarrier (squalenylacetic acid), a high drugloading of 37% was reached. The design of the SQAd prodrug wasintended to protect the fragile adenosine from metabolization,

1Institut Galien Paris-Sud UMR CNRS 8612, Faculty of Pharmacy, University of Paris-Sud XI, 92296 Châtenay-Malabry, France. 2Labex d’ExcellenceNanoSaclay. 3Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara 06100, Turkey. 4Department of Pharmaceutical Technology,Faculty of Pharmacy, Hacettepe University, Ankara 06100, Turkey. 5Department of Neurosurgery, Ankara Ataturk Research & Education Hospital, 06800Bilkent Ankara, Turkey. 6Department of Anatomy, Faculty of Medicine, Hacettepe University, Ankara 06100, Turkey. 7CEA Saclay, iBiTecS-S/SCBM, LabexLERMIT, 91191 Gif-sur-Yvette, France. 8NanoBioPhotonics, Institut d’Electronique Fondamentale, University of Paris-Sud XI, 91405, Orsay Cedex, France.9EA3544, Faculty of Pharmacy, University of Paris-Sud XI, 92296 Châtenay-Malabry, France. 10Institut d’Innovation Thérapeutique, IFR141 ITFM, Faculty ofPharmacy, University of Paris-Sud XI, 92296 Châtenay-Malabry, France. *e-mail: patrick.couvreur@u-psud.fr; karine.andrieux@parisdescartes.fr

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with the subsequent formation of nanoassemblies allowing anaqueous suspension suitable for intravenous administration to beobtained. Transmission electron microscopy (TEM) images ofSQAd nanoassemblies (Fig. 1b) showed monodisperse nano-particles with a mean diameter of ∼120 nm, as also confirmed bydynamic light scattering (DLS) (Fig. 1c, Supplementary Table 1).The average surface charge of the nanoassemblies was found to beapproximately –25 mV, ensuring colloidal stability for at least onemonth when kept at 4 °C (Supplementary Fig. 2). The SQAdnanoassemblies were then fluorescently labelled with CholEsterylBODIPY FL C12 (Supplementary Fig. 3). It was observed thatentrapment of the probe inside the nanoassemblies did not signifi-cantly modify its optical properties (Supplementary Fig. 4), whereasa 1:100 (wt:wt) ratio allowed the best emission signal to be obtainedby avoiding any self-quenching of the fluorophore or eximer for-mation24 (Supplementary Fig. 5). To allow in vivo tracking of thenanoassemblies, we synthesized radiolabelled SQAd, either byproton exchange on the purine base of the adenosine to obtainSQ-3H-Ad (Supplementary Fig. 6) or by performing the couplingof 14C-squalenylacetic acid with adenosine to obtain 14C-SQAd(Supplementary Fig. 7). Fluorescently and radiolabelled SQAdnanoassemblies were obtained without any significant differencesin size or surface charge compared to non-labelled SQAdnanoassemblies (Supplementary Table 1).

Pharmacological efficiencyAlthough adenosine receptor subtypes in the brain and peripheryplay complicated and sometimes opposing roles in cerebral

ischaemia, central administration or continuous intravenousinfusion of adenosine have been shown to be protective againststroke25–27. However, the side effects offset any potential improve-ment in penumbral residual blood flow28,29. Hence, any modifi-cation in adenosine delivery that may alleviate adverse reactionswithout losing its action on the cerebral microcirculation mayprove to be neuroprotective. In this study, we found that intravenousbolus administration of SQAd nanoassemblies dose-dependentlydecreased the infarct volume in mice subjected to 2 h of middlecerebral artery occlusion (MCAo) and 22 h of reperfusion. Pre-ischaemic injection of 7.5 or 15 mg kg−1 SQAd nanoassemblies(equivalent respectively to 2.75 and 5.5 mg kg−1 of adenosine)significantly reduced the stroke size to 31 ± 2 and 17 ± 1 mm3,respectively, compared to the control groups treated with dextrose5% (49 ± 1 mm3), free adenosine (5.5 mg kg−1, 55 ± 3 mm3) orwith unconjugated squalenoyl (SQ) nanoassemblies (9.45 mg kg−1,44 ± 2 mm3) (Fig. 2a). Considering the extremely short plasmahalf-life of adenosine, we tested whether longer-lasting adenosinereceptor agonists could exert neuroprotection comparable tonanoassemblies. Systemic administration of A2 receptor agonist(CGS21680) was not effective, whereas mice treated with A1 recep-tor agonist (CCPA) did not live for 24 h because of cardiac adverseeffects (Supplementary Section I.4). To test if the neuroprotectiveeffect observed with pre-ischaemic administration of SQAd nano-assemblies may have clinical utility, SQAd nanoassemblies(15 mg kg−1) were also administered at reperfusion, 2 h afterstroke. The infarct volume (23 ± 2 mm3) was significantly smallercompared to the controls (Fig. 2a). To evaluate if this neuroprotective

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effect was preserved in the absence of reperfusion, a commonclinical setting30, mice were subjected to 24 h of permanentMCAo. SQAd nanoassemblies (15 mg kg−1) administered 2 h afterthe induction of ischaemia significantly decreased the infarctvolume (24 ± 4 mm3) compared to the vehicle-treated controlgroup (54 ± 3 mm3) (Fig. 2a). Improvements in neurologic deficitscores paralleled the reductions in infarct size in all groups(Fig. 2c). Similar to the decrease in infarct volume (64%), the celldeath processes were also slowed down in the ischaemic areasdestined to infarct in the SQAd nanoassemblies treated group

(15 mg kg−1, pre-ischaemia), as shown by significant reductions at24 h in the number of cells exhibiting apoptotic nuclei withclumped chromatin (Supplementary Fig. 8) or caspase-3 activation(Supplementary Fig. 9). Given the vasodilatory properties of adeno-sine, we investigated if the protective action of SQAd nanoassem-blies could result from a primary vascular mechanism leading to asecondary parenchymal neuroprotection. We observed that mostof the ischaemic microvessels were patent 6 h after re-opening ofthe MCA in SQAd nanoassemblies administered mice, in contrastto untreated animals in whom ischaemic capillaries were still

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Figure 2 | Systemic administration of SQAd nanoassemblies (NAs) provides significant neuroprotection in a mouse model of cerebral ischaemia.a, Ischaemic volumes in control and treated mice subjected to transient (2 h MCAo and 22 h reperfusion) and permanent (24 h MCAo) focal cerebralischaemia were identified by reduced Nissl staining under a light microscope (magnification ×10, insets) (data are presented as mean (mm3) ± s.d., N = 6animals per group; † and * indicate P < 0.05 compared to respective controls). Intravenous administration of 7.5 mg kg−1 or 15 mg kg−1 SQAd nanoassembliesjust before ischaemia or 2 h post-ischaemia significantly decreased the infarct volume compared with control groups that received vehicle (dextrose 5%),adenosine-unconjugated SQ nanoassemblies (9.45 mg kg−1) or free adenosine (5.5 mg kg−1). A significant therapeutic effect was also observed when SQAdnanoassemblies were administered 2 h post-ischaemia in the permanent MCAo model. b, MCAo was performed in mice under anaesthesia by theintraluminal filament method. A filament was advanced in the internal carotid artery through a small incision proximal to the bifurcation up to the origin ofthe MCA to interrupt blood flow to the MCA territory. c, The significant neuroprotective effect of SQAd nanoassemblies was also evident in neurologicevaluation (data presented as mean ± s.d., N = 6 animals per group; † and * indicate P < 0.05 compared to respective controls). Neurologic deficit scores,assessed 24 h after the induction of stroke, paralleled the changes in the infarct volume, although the decreases in deficit scores were less striking, possiblybecause some pyramidal fibres descending from the recovered cortex could not escape damage in the ischaemic subcortical area. d,e, In untreated mice,capillaries in the ischaemic brain were filled with trapped erythrocytes (d, red fluorescence, arrowheads) 6 h after re-opening of the MCA following 2 h ofocclusion, whereas the majority of capillaries were not clogged in SQAd nanoassemblies-treated mice (e). The treatment also reduced ischaemic tissuechanges such as swelling of axon bundles (e, asterisk). f,g, Similarly, capillary structure was well preserved in treated animals (g), unlike controls (f), inwhich astrocyte endfeet ensheathing capillaries as well as endothelial nuclei were swollen (arrowheads) in addition to oedematous neuropil and severaldark-stained degenerating cells surrounded by swollen endfeet (f, asterisks). In treated mice, parenchymal ischaemic changes were mild, limited to lightswelling and scattered degenerating neurons (g, asterisks).

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clogged with trapped erythrocytes (Fig. 2d,e, Supplementary Fig. 10),possibly due to ischaemia-induced pericyte contractions, as previouslyreported31. In addition, astrocyte endfeet around microvessels andendothelial nuclei were not swollen in treated animals, unlike untreatedmice (Fig. 2f,g). The infarct area was significantly reduced in SQAdnanoassemblies treated mice compared to the untreated controls after6 hof reperfusion (SupplementaryFig. 11), that is before significant acti-vation of inflammatory and astrogliotic mechanisms. These findingsindicate that adenosine dissociated from nanoassemblies penetratinginto the endothelium may interact with its receptors on pericytes ofthe cerebral microvessels and astrocytes endfeet, thereby improvingmicrocirculation32.Moreover, adenosine has been shown to be cytopro-tective in low intracellular adenosine triphosphate (ATP) states by pro-moting ATP generation independently of adenosine receptors, whichmay have prevented the observed swelling in the endotheliumand astrocyte endfeet33. It is noteworthy that the concentration ofadenosine achieved within the capillary wall around the neurovascularunit appears to be sufficient to mitigate microcirculation,without inducing systemic vasodilation and resulting hypotension(Supplementary Table 2). In the case of permanent MCAo, adenosinemay promote microcirculatory perfusion in the periphery of the MCAarea, supported by collateral flow with a similar mechanism.

The promising results obtained in the cerebral ischaemiamodel triggered our curiosity regarding further extending the

pharmacological application of SQAd nanoassemblies to the treat-ment of traumatic injury of the spinal cord. It has been shownthat adenosine could provide neuroprotection after spinal trauma,especially through peripheral and central effects on its A2A recep-tors34,35. To determine whether the SQAd nanoassemblies couldimprove functional outcome in a SCI model, we performed a behav-ioural and ultrastructural study in Sprague–Dawley rats undergoinga T9 contusion injury, which is the most relevant model of humanSCI36. Within 5 min post-injury, we injected intravenously eitherSQAd nanoassemblies (32 mg kg−1; equivalent dose of 11.5 mg kg−1

adenosine and 20.2 mg kg−1 squalene), SQ nanoassemblies(20.5 mg kg−1), free adenosine (11.5 mg kg−1) or a solution of dex-trose 5% as control. Functional evaluations were performed37 at 24,48 and 72 h post-injury, and then regularly up to 28 days post-injury. The scores of animals in all groups are presented in Fig. 3band Supplementary Table 3 (see also Supplementary movie). At72 h after trauma, animals injected post-injury with either dextrose5% solution or free adenosine solution (Supplementary Fig. 12a,band Supplementary Movie) were unable to voluntarily move theirhindlimbs, in accordance with the presence of the important trau-matic area observed on the cords (Fig. 3c,d). In contrast, animalsinjected with the SQAd nanoassemblies did not present anyvisible traumatic area on their cords (Fig. 3e) and were fully ableto walk with consistent coordination between the hindlimbs and

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Figure 3 | Pharmacological efficiency of the SQAd nanoassemblies (NAs) in a model of spinal cord injury in rats. a, The pharmacological efficiency of theSQAd nanoassemblies was assessed in a T9 contusion SCI model. Within 5 min, the animals were intravenously injected with either dextrose 5%, SQAdnanoassemblies (32 mg kg−1, equivalent adenosine 11.5mg kg−1, N = 10 animals), SQ nanoassemblies (20.2 mg kg−1, N = 10 animals) or free adenosine(11.5 mg kg−1, N = 10 animals). b, After 24 h, 48 h and 72 h and up to 28 days post-trauma, the animals were functionally graded using the Basso, Beattie andBresnahan grading scale (data presented as mean ± s.e.m., #, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). c–e, After 72 h, the SQAdnanoassemblies-injected animals showed a complete recovery of their hindlimbs, in accordance with the absence of visible traumatic area on the cord (e)compared with the trauma group (c) and the adenosine treated group (d). f–i, After 48 h, the ultrastructure of the myelin configuration of the axons wasstudied by TEM. For animals treated with the solution of free adenosine (g) or only squalene nanoassemblies (h), the small, medium and large myelinatedaxons presented separations and/or interruptions in their myelin configuration (red arrows) compared with healthy animals (f). For SQAd nanoassemblies-injected animals (i), examination of the white matter showed medium and large myelinated axons with separations in the myelin configuration, with a fewof the medium and most of the small myelinated axons found to be ultrastructurally normal (blue arrows). m, myelin; s, myelinated axons with separation inmyelin configuration. j, Quantification of damage on the small myelinated axons showed that the SQAd nanoassemblies dramatically reduced the damagescore (data are presented as mean ± s.d., ****P < 0.0001) compared to all groups.

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forelimbs (Supplementary Fig. 12c–f and Supplementary Movie). Assoon as 9 days after trauma, SQAd nanoassemblies treated animalsachieved a score of 14.4 (Fig. 3b), which is widely accepted as prom-ising and clinically meaningful36 as it is a sign of axonal transduc-tion through the lesion site38. All other treatment groups did notexceed a score of 5 until 28 days post-trauma (Fig. 3b). Because ithas been shown that the preservation of only 5–10% of axons ineach individual tract is sufficient to achieve meaningful locomotorrecovery1, we further investigated the ultrastructure of the myeli-nated axons 2 and 28 days post-injury, using TEM (Fig. 3f–i).Examination of the white matter of control animals (SQ nanoassem-blies or free adenosine solution) revealed that the small, mediumand large myelinated axons had interruptions inmyelin configuration(Fig. 3g,h). In the case of animals treated with the SQAd nanoassem-blies, axons with separations in myelin configuration were detected,but most of these neurons were medium or large in size, with a fewof the medium and most of the small myelinated axons being

normal (Fig. 3i). Quantification of these observations showedthat the SQAd nanoassemblies treatment significantly decreaseddamage to the small (Fig. 3j) and medium (SupplementaryFig. 13) myelinated axons and the swelling of mitochondria(Supplementary Fig. 14), as soon as 2 days post-trauma.

Side effects and systemic toxicityThe emergence of side effects is an important limitation for the useof most CNS medicines39. As adenosine is known to be a neuro-modulator involved in numerous neurological processes13, its thera-peutic use may be associated with the appearance of side effects suchas loss of appetite17,40 or alteration of the sleep cycle18,41. We mon-itored the effect of SQAd nanoassemblies on food intake and bodyweight. Intravenous administration of the SQAd nanoassemblies(15 mg kg−1) did not induce any modification, either in daily foodintake behaviour (Fig. 4a) or in weight variations (SupplementaryFig. 15) of the animals, compared to the controls (dextrose 5%).

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Figure 4 | Absence of side effects and systemic toxicity following intravenous injection of SQAd nanoassemblies (NAs). a,b, The effect of SQAdnanoassemblies on food intake and sleep cycle was assessed in vivo after intravenous injection of either SQAd nanoassemblies (15 mg kg−1) or dextrose 5%.Daily food intake of SQAd nanoassemblies-treated animals was not modified compared with controls (a, black arrow indicates the day of SQAdnanoassemblies or dextrose 5% administration; data presented as mean (g day−1) ± s.d., N = 15 animals per group). The intravenous injection of SQAdnanoassemblies did not induce any modification in the REM sleep quantities (b, data presented as mean (min) ± s.e.m., N = 15 animals per group; insetshows the alternation of dark and light phases in the recording wardrobe). c, To assess if the SQAd nanoassemblies could reproduce the sleeping trigger ofadenosine, the sleep latencies to NREM and REM sleeps were compared and no differences were observed when compared with dextrose 5% controls (datapresented as mean (min) ± s.e.m., N = 15 animals per group). Hence, the intravenous injection of SQAd nanoassemblies prevents the appearance of any sideeffects on the sleep cycle. d–g, The acute (day 1 and day 7) and long-term (day 28) toxicity of SQAd nanoassemblies upon systemic administration wereassessed. SQAd administration did not induce any increase or decrease in the white (d, data presented as mean ×103/μl ± s.d., N = 5 animals per group) orred (e, data presented as mean ×106/μl ± s.d., N = 5 animals per group) blood cell counts. Similarly, no differences in aspartate aminotransferase (AST, f,data presented as mean UI/l ± s.d., N = 5 animals per group) and creatinine (g, data presented as mean mg/dl ± s.d., N = 5 animals per group) were observedbetween the animals injected with SQAd nanoassemblies and the controls (dextrose 5%). WBC, white blood cell; RBC, red blood cell. h–o, Histologicalanalysis of kidneys (h,i), liver (j,k), spleen (l,m) and lungs (n,o), 24 h (h,j,l,n) and 28 days (i,k,m,o) after SQAd nanoassemblies intravenous administration(15 mg kg−1), showed no signs of cell or tissue damage. In each panel, the insets present ×5 magnifications of the areas outlined by black squares.

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The first nocturnal food intake following injection of the treatedanimals was not significantly modified (Supplementary Fig. 16),showing no acute effect of the nanoassemblies. As for the possibleimpact of SQAd nanoassemblies on sleep cycle, it was observedthat the administration of SQAd nanoassemblies did not modifyeither the sleep quantities of non rapid eye movement (NREM)sleep (Fig. 4b, Supplementary Fig. 17a) or rapid eye movement(REM) sleep (Supplementary Fig. 17b), nor the wake quantity(Supplementary Fig. 18) of the treated mice compared to controls.As accumulation of adenosine in the extracellular space of thebasal forebrain during waking has been shown to be a triggeringfactor for sleep42, we also compared the sleep latencies to NREMand REM sleeps. It was observed that the administration of SQAdnanoassemblies did not significantly modify either of these latencies(Fig. 4c), nor the electroencephalogram power spectra(Supplementary Table 4). These results confirm that the dramaticpharmacological efficiency of SQAd nanoassemblies was obtainedwithout triggering any side effect commonly associated withcentral administration of adenosine.

We then investigated the overall toxicity of intravenously admi-nistered SQAd nanoassemblies (15 mg kg−1; 1, 7 and 28 dayspost-injection) to mice, compared to control animals (dextrose5%). No differences were noted between the groups in terms ofwhite blood cell count (Fig. 4d), red blood cell count (Fig. 4e)and other haematological parameters (Supplementary Fig. 19).The levels of aspartate aminotransferase (Fig. 4f), alanine amino-transferase (Supplementary Fig. 20a), creatinine (Fig. 4g) and urea(Supplementary Fig. 20b) were not increased in the SQAd nano-assemblies group, indicating no damage to the kidneys or liver.The observations of kidneys, liver and spleen showed no morpho-logical damages after SQAd nanoassemblies administration, 24 hand 28 days post-injection (Fig. 4h–m). We did not observe anynanoassemblies in the pulmonary territory, with no signs of alveolarobstruction or inflammation (Fig. 4n,o). Finally, using 2D immuno-electrophoresis we showed that the SQAd nanoassemblies led toonly a slight complement activation in human plasma(Supplementary Fig. 21). Together, these results show that theSQAd nanoassemblies may be considered as safe upon systemicintravenous administration at the therapeutic dose of 15 mg kg−1.

Plasma reservoir effectWe further investigated the mechanism behind the observed pharma-cological activity of SQAd nanoassemblies by performing biodistribu-tion studies using two types of radiolabelled nanoassemblies: one with14C labelling located on the squalene moiety and the other with 3Hlabelling on the adenosine moiety (Supplementary Sections I.2 andI.3). In the brain, although 3H could be detected, the lack of 14Cclearly demonstrated the absence of nanoparticle penetration(Supplementary Fig. 22). Additional assays using high-performanceliquid chromatography with radioactive detection (radio-HPLC)also confirmed the absence of either SQAd or adenosine in thebrain after injection of 3H-labelled SQAd (15 mg kg−1)(Supplementary Fig. 23b). As expected, adenosine was not detectedin the brain 1 h following administration of free 3H-adenosine(Supplementary Fig. 23c). These data clearly show that the SQAdnanoassemblies efficacy could not be explained by a central pharma-cological activity, but rather by a primary vascular mechanism leadingto a secondary parenchymal neuroprotection, as already indicated bythe improved microcirculation in the stroke study (Fig. 2d,e). Thisalso suggested that the effectiveness of SQAd nanoassemblies inboth SCI and brain ischaemia could result from a multitargeted pleio-tropic action5 on the cells of the neurovascular unit or other periph-eral cell types34,43, thanks to a sustained systemic circulation.Therefore, the colloidal stability of the SQAd nanoassemblies wasfirst analysed in mouse plasma. Apart from a slight increase thatwas probably due to the formation of a protein corona at their

surface, the mean size of the nanoassemblies did not vary (Fig. 5a,Supplementary Fig. 24), suggesting a good colloidal stability in theperipheral compartment for at least 2 h. The chemical stability ofSQAd was further explored after incubation in mouse plasma andit was clearly observed that SQAd nanoassemblies provided sustainedprotection of the prodrug towards enzymatic degradation (Fig. 5b).Finally, the stability of SQ-3H-Ad after intravenous administrationof the nanoassemblies was assessed by radio-HPLC. We observedthat a significant amount of SQAd remained intact in the blood foras long as 1 h post-injection (Fig. 5c, Supplementary Fig. 25 and 26)

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whereas nothing was detected even 1 min post-injection of3H-adenosine free (Supplementary Fig. 27).

As a whole, these data show that the linkage of adenosine tosqualene and the assembly of the bioconjugate as nanoparticles pro-vides a dramatic protection of adenosine from metabolization bothin vitro after incubation with mouse plasma, and in vivo after intra-venous injection. Thus, SQAd nanoassemblies represent a reservoirof adenosine in the bloodstream that could facilitate delivery toendothelial cells and further cerebrovascular protection. Therefore,the ability of SQAd nanoassemblies to interact with endothelialcells was investigated in vitro using the hCMEC/D3 human cerebralmicrovascular endothelial cell model44. Using flow cytometry andconfocal microscopy, we observed that those cells were able tointernalize the SQAd nanoassemblies (Fig. 6a–h; SupplementaryFig. 28), as was also observed using radiolabelled nanoassemblies(Supplementary Fig. 29). By labelling SQAd nanoassemblies withtwo fluorophores displaying Förster resonance energy transfer(FRET) (Supplementary Section I.4), it was possible to distinguishbetween intact nanoassemblies (presence of a FRET signal) and dis-assembled nanoassemblies (decrease of the FRET signal) after cellcapture. Quantification of the FRET signal by flow cytometryshowed that SQAd nanoassemblies were taken intact by the endo-thelial cells during the first 6 h of incubation (Fig. 6i), before thenanoassemblies progressively dissociated over time inside the

cells, confirming previous in vivo observations (SupplementaryFig. 23) showing that intact SQAd nanoassemblies were unable toenter the CNS.

In this Article, we show for the first time that the linkage of ade-nosine to squalene and subsequent construction as nanoassembliesallow the efficient administration of this molecule with significantpharmacological activity in a brain ischaemia and a SCI model.The demonstrated plasma reservoir effect of SQAd nanoassembliesallowed an improvement of the microcirculation, leading to a sec-ondary parenchyma neuroprotection. Although further studies areneeded to more precisely describe the exact therapeutic mechanismand to determine how dosage, administration frequency and timingof treatment with SQAd nanoassemblies may affect the clinicaloutcome, this study opens a new exciting perspective for the treat-ment of severe neurological diseases where tissue ischaemiaand/or trauma are involved.

MethodsSQAd nanoassemblies. SQAd nanoassemblies were prepared using thenanoprecipitation technique. Briefly, SQAd was dissolved in absolute ethanol, andadded dropwise under strong stearing to a 5% (wt/vol) dextrose solution. Ethanolwas then completely evaporated to obtain an aqueous suspension of pure SQAdnanoassemblies. Fluorescent nanoassemblies were obtained by the same procedure,except 1% (wt/wt) of the fluorescent probe CholEsteryl BODIPY FL C12 was addedto the ethanolic phase. Similarly, radiolabelled nanoassemblies were obtained by

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Figure 6 | Internalization of the SQAd nanoassemblies (NAs) by human cerebral endothelial cells (hCMEC/D3). a, The cell capture of SQAdnanoassemblies by hCMEC/D3 cells was quantified by flow cytometry and compared to cell internalization of the fluorescent probe alone (N = 3independent experiments run in duplicate, data presented as mean (mean fluorescence intensity, MFI) ± s.e.m., **P < 0.01, ***P <0.001). b–g, The effectiveinternalization of the nanoassemblies was confirmed by confocal microscopy after 2 h (b), 4 h (c), 6 h (d), 24 h (e), 48 h (f) and 72 h (g) of incubation.h, It was observed that the nanoassemblies mainly localized in the cell cytoplasm using markers for the nucleus (blue, SYTO61) and for the cellularmembrane (red, WGA AlexaFluor 555). i, Internalization of FRET SQAd nanoassemblies showed that the nanoassemblies disassembled inside the cells after6 h of incubation (data presented as mean (MFI) ± s.e.m., Supplementary Section I.7.).

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adding SQ-3H-Ad or 14C-SQAd to the ethanolic SQAd solution. Nanoassembliessize and surface charge were systematically measured after preparation using aMalvern Nano ZS, and morphology was observed by cryogenic TEM (cryoTEM).

Focal cerebral ischaemia injury model. The proximal MCA was occluded bythe filament method in male SWISS albino mice (28–32 g) under isofluraneanaesthesia. To confirm occlusion and reperfusion, the regional cerebral blood flowwas measured by laser-Doppler flowmetry. The body temperature was kept at37 ± 0.1 °C. Tissue O2 saturation and arterial blood pressure were monitored non-invasively. Mice were randomly allocated to 10 groups and were subjected to 2 hischaemia and 22 h of reperfusion or 24 h of permanent ischaemia (SupplementaryTables 2 and 6). Treatments were administered intravenously just before inducingischaemia or 2 h after ischaemia (N = 6 animals per group, Supplementary Table 6).Mice were blindly neurologically evaluated before being killed. Infarct volumes wereassessed by Nissl staining, caspase-3 activation by immunolabelling for cleavedcaspase-3, and apoptotic nuclear morphology by dark field microscopy. Another setof animals were killed and perfused to flush erythrocytes, 6 h after 2 h MCAo,and entrapped erythrocytes were rendered fluorescent with NaBH4.

SCI model in rats. After general anaesthesia using xylazine and ketaminehydrochloride, Sprague–Dawley rats (200–250 g) underwent a laminectomy and aSCI was produced by dropping a 4 g weight from a height of 12.5 mm. Within 5 minpost-injury, treatments were injected intravenously (N = 10 animals per group,Supplementary Table 8). Functional evaluations were performed blindly 24, 48 and72 h and regularly up to 28 days post-injury using the Basso, Beattie and Bresnahangrading scale. For ultrastructural evaluation, tissue samples were resected from thetraumatic area of the spine 48 h and 28 days post-injury. Tissue samples wereexamined blindly by TEM.

Assessment of the effect on food intake and sleep cycle of SQAd nanoassembliesin mice. A number of 25 g male SWISS albino mice housed in individual cages wereinjected with either SQAd nanoassemblies (15 mg kg−1) or dextrose 5% (N = 10animals per group). Individual mice and their corresponding food were weighed 12h post-injection and then regularly for seven days. For sleep cycle experiments, 25 gmale SWISS albino mice were implanted with two cortical, two ocular and twomuscular electrodes. Seven days post-surgery, they were individually placed inrecording cages and allowed to habituate for three days. On the day of theexperiment, the mice were disconnected, intravenously injected with eitherSQAd nanoassemblies (15 mg kg−1) or dextrose 5% (N = 15 animals per group) andthen immediately reconnected for 24 h recording.

Assessment of the systemic toxicity of SQAd nanoassemblies in mice. Several25 g male SWISS albino mice were injected with either SQAd nanoassemblies(15 mg kg−1) or dextrose 5% (N = 5 animals per group). At 1, 7 or 28 days post-injection, the animals were anaesthetized using pentobarbital and blood wascollected by intracardiac sampling for haematology and serum analysis. Liver,kidneys, spleen and lungs were then excised, fixed, paraffin-embedded, and cut into5-μm-thick sections. Haematoxylin and eosin staining was performed on all theorgans for analysis of the morphology.

Pharmacokinetics of radiolabelled SQAd nanoassemblies in mice. RadiolabelledSQAd nanoassemblies were injected into 25 g male SWISS albino mice (15 mg kg−1,600 µCi kg−1). At 1 min, 5 min, 15 min, 30 min, 1 h and 24 h post-injection,animals were anaesthetized using pentobarbital, and blood was collected byintracardiac sampling and immediately centrifuged to isolate plasma. Samples werethen analysed by radio-HPLC.

Nanoparticle internalization by hCMEC/D3 (flow cytometry and confocalmicroscopy). hCMEC/D3 cells were seeded in 24-well plates (50,000 cells/cm2).Cells were grown for 7 days and then incubated with 10 µg ml−1 fluorescent SQAdnanoassemblies. For flow cytometry, cell fluorescence was recorded using a flowcytometer C6 with an excitation source at 488 nm and emission collection at515 nm. For confocal microscopy, if required at the end of the incubation time,nuclei were stained with 10 µM SYTO61 for 2 h at 37 °C, followed by membranestaining with 10 µg ml−1 WGA AlexaFluor 555 for 20 min at 37 °C. Cells were thenfixed and imaged with a LSM510 Zeiss confocal microscope using lasers at 488 nm(nanoassemblies), 543 nm (membranes) and 633 nm (nuclei).

Received 17 September 2013; accepted 21 October 2014;published online 24 November 2014; corrected after print3 December 2014

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AcknowledgementsThe research leading to these results received funding from the European Research Council(allocated to P.C.), under the European Community’s Seventh Framework ProgrammeFP7/2007-2013 (grant agreement no. 249835). A.G. is supported by a NerF-ENP fellowship

provided by the Région Ile-de-France. T.D.’s work is supported by the Turkish Academy ofSciences. M.Y. is supported by a limited grant by L’Oréal, Turkey. H.E. has been supportedby theHacettepe University Scientific Research Project (project no. 013D04301002). O.T. issupported by the European Union Seventh Framework Programme FP7/2007-2013 (grantagreement no. 246556). The authors thank D. Sobot (Institut Galien Paris-Sud XI) for helpwith flow cytometry experiments, O. Bawa and P. Opolon (Institut Gustave Roussy) foranalysis of the morphology on stained slides, M. Wittner (Institut Gustave Roussy) for thehaematology study, S. Beurlet (VEBIO Lab) for biochemical dosages andM. Parrod (BertinPHARMA) for the radio-HPLC analysis.

Author contributionsP.C. and T.D. conceived and designed the research. A.G. designed and performed thenanoparticle preparation, the side effects and toxicity experiments, the stability and in vivopharmacokinetic/biodistribution studies and the in vitro experiments. S.L. developedand performed the SQAd synthesis, D.D. helped with analysing the chemical results. B.R.,S.G.A., G.P. andO.L. developed and performed the radiolabelled compound synthesis. T.D.and M.Y. designed and performed the cerebral ischaemia experiments. B.D-D. performedthe histological stainings and countings for cerebral ischaemia experiments. S.C. and Y.C.were in charge of nanoparticle preparation for the cerebral ischaemia experiments. H.E.,O.F.T. and A.G. designed and performed the spinal cord injury experiments. M.F.Z.performed the ultrastructural evaluation of the spinal cord injury experiments. A.G., O.T.and N.H designed and performed the FRET nanoassemblies experiments. Y.L.D and A.G.performed the sleep cycle experiments. J.M. performed the HPLC analysis. S.V. performedthe complement activation experiments. H.C. helped to analyse the radioactivity data andV.N. helped to analyse the confocal data. P.C., T.D., K.A., A.G. andM.Y. co-wrote the paper.All authors discussed the results and commented on the manuscript.

Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints. Correspondence andrequests for materials should be addressed to K.A. and P.C.

Competing financial interestsThe authors declare no competing financial interests.

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In the version of this Article originally published, the affiliations of Hakan Eroglu, Omer Faruk Turkoglu, Seçil Caban, Oya Tagit, Niko Hildebrandt and Yilmaz Capan were incorrect; they should have appeared as shown below. This error has now been corrected in the online versions of the Article.

Hakan Eroglu4, Omer Faruk Turkoglu5, Seçil Caban4, Oya Tagit8, Niko Hildebrandt8, Yilmaz Capan4

4Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, Ankara 06100, Turkey.5Department of Neurosurgery, Ankara Ataturk Research & Education Hospital, 06800 Bilkent Ankara, Turkey.8NanoBioPhotonics, Institut d’Electronique Fondamentale, University of Paris-Sud XI, 91405, Orsay Cedex, France.

Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injuryAlice Gaudin, Müge Yemisci, Hakan Eroglu, Sinda Lepetre-Mouelhi, Omer Faruk Turkoglu, Buket Dönmez-Demir, Seçil Caban, Mustafa Fevzi Sargon, Sébastien Garcia-Argote, Grégory Pieters, Olivier Loreau, Bernard Rousseau, Oya Tagit, Niko Hildebrandt, Yannick Le Dantec, Julie Mougin, Sabrina Valetti, Hélène Chacun, Valérie Nicolas, Didier Desmaële, Karine Andrieux, Yilmaz Capan, Turgay Dalkara and Patrick Couvreur

Nature Nanotechnology 9, 1054–1062 (2014); published online 24 November 2014; corrected after print 3 December 2014.

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