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1 | SCIENCE ADVANCES RESEARCH ARTICLE HEALTH AND MEDICINE Copyright © 2019 Authors, some The rights reserved; A new painkiller nanomedicine to bypass the exclusive licensee American Association blood-brain barrier and the use of morphine for the Advancement of Science. No claim to 1 1 1 1 1,2 Gautier , , Simona Mura , Anne , Catherine Cailleau Lepetre-Mouelhi Jiao Feng , Sinda Government original U.S. 1 2 3 Couvreur Coudore * , Michel François Hamon , Patrick Works. Distributed under a Creative The clinical use of endogenous neuropeptides has historically been limited due to pharmacokinetic issues, includ- Commons Attribution ing plasma stability and blood-brain barrier permeability. In this study, we show that the rapidly metabolized NonCommercial enkephalin (LENK) neuropeptide may become pharmacologically efficient owing to a simple conjugation Leu- License 4.0 (CC BY-NC). with the lipid squalene (SQ). The corresponding LENK-SQ bioconjugates were synthesized using different chemical in order to modulate the LENK release after their formulation into nanoparticles. This new SQ-based nano- linkers formulation prevented rapid plasma degradation of LENK and conferred on the released neuropeptide a notable antihyperalgesic effect that lasted longer than after treatment with morphine in a rat model of inflammation (Hargreaves test). The biodistribution study as well as the use of brain-permeant and -impermeant opioid receptor antagonists indicated that LENK-SQ NPs act through peripherally located opioid receptors. This study represents Downloaded from a novel nanomedicine approach, allowing the specific delivery of LENK neuropeptide into inflamed tissues for pain control. To date, the two main approaches to enhancing the analgesic INTRODUCTION Pain represents an important global health challenge for many rea- activity of opioid peptides relied on (i) the increase of the stability of http://advances.sciencemag.org/ sons, including high prevalence, serious associated sequelae, and the endogenous peptides using enkephalinase inhibitors or (ii) the chemi- relative lack of efficient treatment, especially for neuropathic pain cal synthesis of exogenous peptides with enhanced lipophilicity and alleviation. Pain-relevant disorders such as arthritis, cancer, and degradation resistance. However, because of insufficient enzymatic pathological changes in the nervous system are highly prevalent and specificity, the enkephalinase inhibitors are often endowed with 1 bring great inconvenience and distress to the patients ( ). Chronic poorly tolerated side effects ( ). In addition, the derivatization of 9 pain has a significant impact not only on the patients themselves peptides often ends up with biologically inactive compounds, and but also on the broader community and economy. With the acti- the same applies to neuropeptides covalently linked to transport vation of μ-opioid receptors, morphine and the related synthetic 10 ). This explains vectors for crossing the blood-brain barrier (BBB) ( opioids become the most powerful and most widely used painkill- why none of the research efforts performed decades ago has resulted ers in current clinical practice. However, morphinic treatments are in marketed medicines. on May 5, 2019 associated with severe side effects, such as respiratory depression On the other hand, although nanoparticulate drug delivery sys- and addiction linked to the development of opioid tolerance and tems are an efficient approach for protecting drug molecules from 2 ). According to the National Vital Statistics System of dependence ( rapid metabolization, only a few were applied to enkephalins and en- the U.S. Centers for Disease Control and Prevention/National Center ( kephalin derivatives. In a primary study, Kreuter et al. ) showed 11 ), every day, more than 115 people in the 3 for Health Statistics ( that intravenously injected dalargin-loaded polybutylcyanoacrylate United States die after overdosing on opioids. The misuse of and nanoparticles (NPs) coated with a non-ionic surfactant induced addiction to opioids, especially morphine, is a serious national crisis time- and dose-dependent antinociceptive effects. In subsequent in the United States (and probably also in other countries) that affects studies, positively charged nanocarriers were also used for the delivery public health as well as social and economic welfare. This highlights of opiate- – - 12 ). Never 15 related drugs and peptides into the brain ( the need to urgently find new painkillers. In this context, endogenous theless, these approaches have met with limited success because, neuropeptides, such as enkephalin, remain an attractive option. in general, the amount of NPs able to cross the BBB remains  Enkephalins activate both -opioid receptors, but with a - and  very low (less than 1% of the injected dose) ( 16 ). In addition, the  10-fold higher affinity toward ). Compared -opioid receptors ( 4 toxicity and elimination of NPs from the brain parenchyma remain with  -opioid receptor agonists,  -opioid receptor ligands are a major issue; this is especially true for the abovementioned cationic ), as well as reduced 5 believed to have a much lower abuse potential ( nanodrugs. 8 ) impairments. respiratory ( 6 ), gastrointestinal ( 7 ), and cognitive ( Thus, the design of safe analgesic nanoformulations capable of However, enkephalins have historically been limited because of restricting their activity peripherally and optimizing drug concen- pharmacokinetic issues and rapid plasma metabolization. tration at the site of injury may overcome these issues. Another ad- vantage of targeting peripheral opioid receptors is that it prevents and reverses the effects of multiple excitatory agents expressed in 1 ). 17 damaged tissue ( Institut Galien Paris-Sud, UMR8612, Univ. Paris-Sud, Université Paris-Saclay, Châtenay- 2 Malabry 92290, France. Centre de Psychiatrie et Neurosciences, INSERM UMR 894, Here, we report a very simple and easy way to use the currently 3 Université Paris Descartes, 75014 Paris, France. Laboratoire de Neuropharmacologie, unusable Leu-enkephalin (LENK) as an analgesic drug following intra- INSERM UMRS 1178, Univ. Paris-Sud, Université Paris-Saclay, Châtenay-Malabry venous injection. To this goal, a new nanoformulation was achieved, 92290, France. which proved capable of precise and efficient delivery of LENK for *Corresponding author. Email: [email protected] et al 1 of 12 Feng 5 2019; Sci. Adv. ., : eaau5148 13 February 2019

2 | RESEARCH ARTICLE SCIENCE ADVANCES pain control. Practically, LENK was conjugated to squalene (SQ), a during the deprotection step, this approach was abandoned in favor natural and biocompatible lipid, through various chemical linkers, of the Alloc (allyloxycarbonyl) strategy. Thus, LENK was protected resulting in a library of LENK lipidic prodrugs, which allowed the with an Alloc group on its N-terminal amine before reacting controlled release of the peptide. As shown previously with anticancer with the chloromethyl ester of squalenic acid. Subsequent deprotec- compounds ( 18 ), the linkage of LENK with SQ triggered the sponta - tion of Alloc-LENK-SQ under neutral conditions was then achieved neous self-assembly of the bioconjugates into LENK-squalene via the catalytic transfer hydrogenation method using triethylsi- nanoparticles (LENK-SQ NPs) in water, which was attributed to the ), affording pure LENK-SQ-Diox in 19 lane (TES) and 10% Pd-C ( dynamically folded conformation of the natural lipid. The analgesic 9.5% yield. effect of these LENK-SQ NPs was evaluated in a carrageenan- -diglycolate spacer was ′ The LENK-SQ-Dig prodrug with a 2 induced pain model using a thermal nociception test (Hargreaves) synthesized by the reaction of squalenol with diglycolic anhydride to assess hyperalgesia. Pain sensitivity was rated in response to a hot before reaction with the condensing agent and LENK, resulting in stimulus on the inflamed hind paw of rats. In addition, the in vivo 69% yield. The oxa moiety of the linker was intended to enhance the biodistribution of NPs was investigated in mice using in vivo fluo - susceptibility to hydrolysis by increasing the distance between LENK rescence imaging for assessing the ability of the LENK-SQ NPs to and SQ and, thus, the accessibility to the linkage. Direct conjugation target the inflamed tissue. Last, a toxicological study was also per- between LENK and squalenic acid via single amide bond was formed to ensure the safety of these NPs. achieved by acid activation using ethyl chloroformate, affording LENK-SQ-Am in 73% yield. Downloaded from RESULTS Preparation and characterization of LENK-SQ NPs Synthesis of LENK-SQ conjugates All bioconjugates showed the capability to self-assemble as NPs in In this study, various LENK-SQ conjugates were designed with dif- aqueous solution after nanoprecipitation from LENK-SQ ethanolic ferent linkers using bioconjugation (Fig. 1). Conjugation of squalene solutions. When measured by dynamic light scattering (DLS), the to LENK was performed by exploiting two sites: C-terminal acid size of the NPs varied from 60 to 120 nm, depending on the linkage and N-terminal amine. To modulate the release kinetics of LENK between squalene and enkephalin (Fig. 2). The difference in NP zeta http://advances.sciencemag.org/ from NPs, we used three linkers with different sensitivity to hydro- potential was related to the nature of the exposed amino acids onto lysis in the following order: dioxycarbonyl (LENK-SQ-Diox also the NP surface. In the case of the LENK-SQ-Diox bioconjugate, the called “sensitive bound”) > diglycolic (LENK-SQ-Dig) > amide SQ conjugation on the C-terminal LENK peptide let its N-terminal (LENK-SQ-Am). site free (primary amino group), leading to a net positive charge. In Practically, the squalenic acid was coupled to C-terminal LENK contrast, the zeta potential became negative when the conjugation using a dioxycarbonyl linker (LENK-SQ-Diox, conjugate 1) or with SQ was performed on the N-terminal LENK peptide (LENK-SQ- squalenol was conjugated to N-terminal LENK through a diglycolic Dig and LENK-SQ-Am). Drug loadings (Fig. 2) ranged between 53 spacer (LENK-SQ-Dig, conjugate 2). Starting from squalenic acid, it and 60%, which was much higher than in conventional nanoparticles was also possible to perform the linkage to N-terminal LENK using 21 ). Figure 2 , or liposomes, which amounted to a maximum of 5% ( 20 a simple amide bond (LENK-SQ-Am, conjugate 3). shows representative cryogenic transmission electron microscopy on May 5, 2019 The LENK-SQ-Diox conjugate was synthesized by alkylation of (cryo-TEM) images of the LENK-SQ NPs at a concentration of the carboxylate function of the peptide with the chloromethyl ester 4 mg/ml in Milli-Q water. They displayed spherical and mono- of squalenic acid, which was prepared upon treatment of squalenic disperse structures with sizes ranging from 50 to 100 nm. The slight acid with chloromethyl chlorosulfate. To avoid N-terminal conju- discrepancy between DLS and cryo-TEM size measurements could gation, the Fmoc (9-fluorenylmethoxycarbonyl) strategy was first be attributed to the known hydrodynamic radius-related differences adopted for the protection of the primary amino group of LENK, ( ). The sizes and the surface charges of the LENK-SQ NPs were 22 but due to the early release of the peptide from Fmoc-LENK-SQ found to be quite stable at +4°C (fig. S6). Fig. 1. Chemical structures of the bioconjugates. (1) Leu-enkephalin-squalene with dioxycarbonyl linker (LENK-SQ-Diox), (2) Leu-enkephalin-squalene with diglycolic linker (LENK-SQ-Dig), and (3) Leu-enkephalin-squalene with amide linker (LENK-SQ-Am). 2 of 12 et al ., Sci. Adv. 2019; 5 : eaau5148 13 February 2019 Feng

3 | RESEARCH ARTICLE SCIENCE ADVANCES Effect of morphine on thermal hyperalgesia The acute treatment with morphine (1 mg/kg; Fig. 5A) reduced the thermal hyperalgesia, as shown by the resulting significant in- crease in PWLs. Ten minutes after morphine injection, the PWL reached 12.87 ± 1.38 s, while it remained at 3.05 ± 0.20 s after treat- ment with a control dextrose solution (Fig. 5A). However, mor- phine antihyperalgesic pharmacological activity disappeared rapidly, and no significant effect was observed as soon as 100 min after mor- phine administration (Fig. 5A). Effect of LENK-SQ NPs on thermal hyperalgesia We evaluated the antihyperalgesic effect of LENK-SQ NPs with the three different linkers during 4 hours after their administration (Fig. 5, C to H). All injected rats with LENK-SQ NPs displayed sig- Representative cryo-TEM images showing the forma- Fig. 2. NP characterization. nificant reduction of thermal hyperalgesia, as expressed by a marked ) LENK-SQ-Dig A B ) LENK-SQ-Diox NPs, ( tion of NPs from different bioconjugates: ( increase of the respective area under the curve (AUC) values in ) LENK-SQ-Am NPs. Scale bars, nm. NPs, and ( C 100 Physicochemical characteristics comparison with  -carrageenan–treated rats injected with either of NPs [i.e., size, polydispersity index (PDI), zeta potential, and % drug loading] are the free LENK peptide or the blank SQ NPs (Fig. 5, D, F, and H). In shown in the table. particular, the antihyperalgesic activity was significant at all time Downloaded from points from 10 to 130 min in rats injected with LENK-SQ Diox NPs or LENK-SQ Am NPs (Fig. 5, C and G). As shown in Fig. 5E, LENK- In vitro release of LENK from NPs in serum SQ- dig NPs also displayed a significant antihyperalgesic effect, with The incubation of LENK-SQ-Diox in serum resulted in a decrease a maximum increase in PWL maintained from 10 to 130 min after of the bioconjugate, which correlated well with the release of the injection, and a progressive decline down to baseline at 220 min. peptide (Fig. 3A). The concentration of the bioconjugate decreased Maximal PWL values reached after the administration of LENK-SQ gradually till 7 hours, while LENK-SQ-Diox NPs progressively re- http://advances.sciencemag.org/ NPs in -carrageenan–treated rats corresponded to basal PWL val-  leased the free LENK peptide. The peptide was then slowly degrad-  -carrageenan treatment ues measured in control naïve rats, before ed by the peptidases of the serum but still lasted beyond 10 hours (Fig. 5, C, E, and G), indicating a pure antihyperalgesic action of after incubation (Fig. 3A). The incubation of LENK-SQ-Dig in se-  these NPs. In contrast, morphine injection in -carrageenan– treated rum resulted in a decrease of the bioconjugate until complete disap- rats resulted in PWL values twice as high as those found in control pearance at 2 hours, but no presence of free peptide was detected. naïve rats (Fig. 5A), as expected from not only an antihyperalgesic The reverse-phase high-performance liquid chromatography (RP- effect but also the well-established analgesic effect of the opiate HPLC) analyses, however, highlighted a slow release of the peptide agonist. In addition, blank SQ NPs (without the LENK) did not still attached to its linker. This release reached a maximum at demonstrate any antihyperalgesic activity (Fig. 5), which indicated 45 min, followed by progressive degradation of the peptide-linker that the analgesic response to LENK-SQ NP administration resulted fragment that could still be detected over 10 hours (Fig. 3B). In con- on May 5, 2019 from the release of the LENK peptide. trast, LENK-SQ-Am remained stable in serum, without a signifi- Effects of opioid receptor blockade using naloxone and cant decrease for a period of 48 hours, and no peptide was released naloxone methiodide in the course of the experiment (Fig. 3C). It was observed that the To ascertain the involvement of central or peripheral opioid recep- degradation of the free LENK peptide was very fast (half-life, 2 min), tors during the antihyperalgesic effect of LENK-SQ NPs, we sub - whereas the LENK-SQ bioconjugate was unaffected during the course or cutaneously injected naloxone (Nal, a brain-permeant opioid recept of the experiment (60 min) (fig. S7). antagonist) or naloxone methiodide (Nal-M, a brain-impermeant 24 ) 15 min before the injection of mor- opioid receptor antagonist) ( Analgesic efficacy of LENK-SQ NPs phine or NPs (Fig. 4). Preadministration of the nonselective opioid The antihyperalgesic effect of LENK-SQ NPs was determined in a receptor antagonist Nal (0.5 mg/kg subcutaneously) abolished the carrageenan-induced paw edema model in rats. Baseline measure- amplitude and the duration of the antihyperalgesic effect of mor- ments of paw withdrawal latencies (PWLs) were made before carra- phine (PWL, 3.20 ± 0.59 s versus 12.87 ± 1.38 s at 10 min) and de- ) and presented a geenan injection using the Hargreaves test ( 23 creased the corresponding AUC value by 81% in comparison with mean value ± SEM ( = 8) of 6.65 ± 0.37 s. Then, thermal sensitivi- n the morphine group (Fig. 5B). The peripheral opioid receptor an- ties were evaluated 3 hours after carrageenan injection into the right tagonist, Nal-M, was markedly less effective since it reduced the hind paw, which corresponded to the peak inflammatory response. morphine’s effect by only 13% (Fig. 5, A and B). Preadministration We assessed antihyperalgesic effects using the same test at various of either Nal or its quaternary derivative Nal-M abrogated the anti- times after acute administration of the different drug treatments at hyperalgesic effect of the three LENK-SQ NPs (Fig. 5, C to H). Nal this 3-hour inflammation peak (Fig. 4). pretreatment caused a reduction of 66, 105, or 73% AUC values Effect of intraplantar  -carrageenan injection on thermal sensitivity compared to these found in rats injected with LENK-SQ-Diox, Intraplantar injection of  -carrageenan into the right hind paw in- LENK-SQ-Dig, and LENK-SQ-Am NPs alone, respectively. The duced a local inflammatory response characterized by marked edema, corresponding reductions in AUC values with Nal-M reached 81, hyperthermia, and hyperalgesia restricted to the injected right hind 99, and 96%, respectively, indicating that the selective blockade of paw. Thermal hypersensitivity was developed in all the rats, with only the peripheral opioid receptors was enough to abrogate the an- a mean decrease of 52.48% PWL compared to the basal PWLs in tihyperalgesic effects of LENK-SQ NPs. naïve rats. ( P < 0.001; see Fig. 5). : eaau5148 13 February 2019 5 2019; Sci. Adv. ., 3 of 12 et al Feng

4 | SCIENCE ADVANCES RESEARCH ARTICLE ) LENK-SQ with diglycolic Fig. 3. In vitro bioconversion of LENK-SQ bioconjugates into LENK in the presence of serum. ) LENK-SQ with dioxycarbonyl linker, ( ( A B linker, and ( C ) LENK-SQ with amide bond. Solid lines and dashed lines represent the bioconjugates and the released peptides, respectively. Biodistribution of LENK-SQ NPs The levels of aspartate transaminase (AST; fig. S10A) and alanine Downloaded from We assessed the in vivo biodistribution of LENK-SQ-Am NPs after transaminase (ALT; fig. S10B) were not increased in the LENK-SQ ′ - ′ ′ intravenous injection of fluorescent DiD (1,1 ,3 -dioctadecyl-3,3,3 NP group, indicating no toxicity toward the liver, which was con- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt)–labeled firmed by histological analysis of this tissue at 24 and 48 hours -carrageenan–induced paw edema LENK-SQ-Am NPs in a murine  (fig. S10, C to F). The observations of the spleen (fig. S10, G to J), the model (right hind paw). The fluorescence in tissues was monitored kidneys (fig. S10, K to N), the lungs (fig. S10, O to R), and the heart up to 24 hours, noninvasively, from the abdomen side using an IVIS (fig. S10, S to V) did not show any morphological damage after http://advances.sciencemag.org/ Lumina (Fig. 6). Mice injected with saline into the paw were used as LENK-SQ NP administration either. Together, these results show non-inflamed control. The real-time in vivo imaging showed, in that the LENK-SQ NPs may be considered as safe upon systemic comparison with the healthy paw, an increase of two to three times that intravenous administration at the therapeutic dose of 20 mg/kg. of the average radiant efficiency within the inflamed paw after intra- venous injection of fluorescent LENK-SQ-Am NPs (Fig. 6, A, D, and F). DISCUSSION  -carrageenan–administered mice In a control experiment, when the Peptides and proteins have great potential as therapeutic macro- were intravenously injected with a single DiD solution, no significant molecules. However, their use in clinical practice is generally ham- accumulation of fluorescence was observed in the inflamed paw (Fig. 6, ). In this 25 pered by poor serum stability and rapid metabolization ( C and F). In another control experiment, mice were injected locally context, the “squalenoylation” technology should be of great interest, with saline in the hind paw and intravenously treated with fluorescent on May 5, 2019 as we showed here that it allows the delivery of therapeutic amounts LENK-SQ NPs. No significant accumulation of fluorescence at the of LENK neuropeptide for efficient pain control. The so-called hind paw level was observed under this condition (Fig. 6, B and E), squalenoylation approach, which refers to the linkage of a drug to -carrageenan–  showing that the accumulation of fluorescence in the the squalene, has already been proposed for small molecules, mainly inflamed paw was not due to the local hind paw injection per se. In a n for anticancer compounds, such as gemcitabine, doxorubicin, or additional experiment, it was shown that the incubation in serum of cisplatin ( ), or for other molecules, such as adenosine, ddI, or 26 LENK-SQ NPs containing the fluorescent dye (DiD) and a fluorescence ddC ( 27 ). Nevertheless, the conjugation of a peptide to SQ has been quencher [DiR (1,1′ -dioctadecyltetramethyl indotricarbocyanine an innovative but tricky achievement for the following reasons: (i) iodide)] resulted in the progressive appearance of fluorescence, the Peptides are unstable biomolecules, and their chemical engineering dynamics of which indicate a relatively slow dissociation of LENK-SQ is not easy, and (ii) peptides are hydrophilic molecules, whereas NPs in serum: 20% after 5 min and 50% after 30 min. This suggested SQ is a lipid, insoluble in water. This makes the chemical reaction that, under our in vivo conditions, a significant proportion of intact rather uncertain, and (iii) the chemical modification of a peptide often NPs could reach the inflamed tissue (fig. S8). results in a loss of its pharmacological activity. The chemical approach Last, in a separate experiment, 4 hours after the intravenous in- used in the present study has overcome all these complications, al- jection of fluorescent NPs or DiD solution, animals were eutha- lowing the design of a small library of innovative enkephalin- nized and transcardially perfused with 40 ml of saline to remove the squalene bioconjugates with a preserved pharmacological activity. fluorescence from the blood. After collection of tissues, a strong For the synthesis of these bioconjugates, we took advantage of the ex vivo fluorescence signal was again observed not only in the in- remarkable, dynamically folded conformation of SQ to chemically flamed paw but also in the liver, the spleen, and the lungs, whereas conjugate this natural lipid with the neuropeptide using different no detectable accumulation of fluorescence occurred in the brain of linkers. Although it is well known that the N terminus of LENK the animals (fig. S9). is required for binding to opioid receptors ( 28 ), the conjugation on N terminus was also achieved, based on the fact that the amide Toxicity study bond is susceptible to be cleaved by overexpressed peptidases with- The overall toxicity of LENK-SQ NPs was investigated 24 and ). Thus, we synthesized the resulting 29 in an inflammation site ( 48 hours after their intravenous administration (20 mg/kg) in rats LENK-SQ prodrugs with either direct amide bond or diglycolic or and compared to control animals injected with 5% dextrose solution. et al Feng : eaau5148 13 February 2019 5 2019; Sci. Adv. ., 4 of 12

5 | RESEARCH ARTICLE SCIENCE ADVANCES Fig. 4. Experimental design for algesimetry. The antinociceptive effect of NPs was tested in a pathophysiological context induced by an intraplantar carrageenan injection (2% saline, 100  l). Involvement of central or peripheral opioid receptors was performed using a brain-permeant opioid antagonist, naloxone (Nal ), and a brain- impermeant opioid receptor antagonist, naloxone methiodide (Nal-M). NP suspensions or control solutions were injected intravenously with a dose volume of 10 s . uring 30 ml/kg d The Hargreaves test was performed 10 min after the NP administration and then every 30 ent mg/kg) was equival min. The dose of LENK-SQ NPs (20 min up to a period of 250 to LENK (11.48 mg/kg) and corresponded to 20.66 mg/kg) and to SQ NPs (8.28 mmol/kg for both LENK-SQ and LENK. s.c., subcutaneous; i.pl., intraplantar. Downloaded from dioxycarbonyl linker to investigate the possible influence of the SQ moiety. The absence of release of LENK from LENK-SQ-Am linkage stability on the peptide release. It was expected that LENK- was expected given that, in general, an amide bond is chemically SQ-Diox released faster than LENK-SQ-Dig, and LENK-SQ-Am was , 32 ). Further- 31 and enzymatically more stable than an ester bond ( supposed to trigger the slower release as reported in the literature more, it is well known that N-terminal modification of linear ( 30 ). Both LENK-SQ-Am and LENK-SQ-Dig were obtained in peptides increases the peptidase resistance, which is the case for http://advances.sciencemag.org/ good yields (around 70%). The third one with dioxycarbonyl linker ). Last, as mouse serum is LENK-SQ-Am and LENK-SQ-Dig ( 33 led to a lower yield due to an additional step for removal of the Alloc particularly rich in esterases, it was expected that the release of group, followed by two successive purifications. LENK from LENK-SQ NPs mainly resulted from enzymatic hydro- The LENK-SQ bioconjugates were then formulated as nanopar- lysis of the ester bond ( ). All three conjugates were then recruited 31 ticles in dextrose solution (2 mg/ml) with impressively high drug for antihyperalgesia experiments. It was expected that the more payload (i.e., 53 to 59%) using a simple nanoprecipitation technique aggressive in vivo enzymatic content, particularly rich in proteolytic without the aid of any surfactant. It is noteworthy that this drug enzymes at the inflammation site, will contribute to the release payload was markedly higher than that in liposomes ( 20 ) or PLGA 29 ). The presence of high con- of LENK from all the bioconjugates ( 21 -glycolic acid)] ( co [poly(lactic- ) enkephalin-loaded nanoparticles centrations of proteolytic enzymes such as chymotrypsin, cathepsin (0.4 and 4.75%, respectively, drug loading), which might explain why D, and other proteases in inflammatory exudates has been reported, on May 5, 2019 - the latter two nanoformulations had never been used for in vivo pharma which also indicates their important role in the inflammatory cological studies. The size of the NPs varied from 61 to 112 nm, de- process ( 29 ). pending on the peptide and the conjugation site (Fig. 2). The NPs Then, the antihyperalgesic properties of LENK-SQ NPs were displayed spherical and monodisperse structures with net positive or evaluated using an animal model of inflammatory hyperalgesia that negative surface charge (Fig. 2), which could be attributed to the free 34 ). We assessed the anti- mimics human clinical pain conditions ( terminal function of the peptide depending on the bioconjugation hyperalgesic activity after a single intravenous administration of the mode. Free N-terminal amine function led to a net positive surface different LENK-SQ NPs in the -carrageenan–induced inflamma-  charge, while free C-terminal acid function resulted in a net negative tory paw model using the Hargreaves test. All LENK-SQ NPs dis- surface charge. played a significant antihyperalgesic effect on inflamed hind paw. To ascertain that the free LENK peptide could be released from The antihyperalgesic effect was less intense than after morphine the LENK-SQ NPs, we tested the chemical stability of the different treatment, but NPs evidenced a much longer-lasting effect. Un- linkers (i.e., direct amide or dioxycarbonyl or diglycolate spacers) expectedly, LENK-SQ-Am NPs, which was expected to release the after incubation of the LENK-SQ NPs with mouse serum (Fig. 3). peptide slower in comparison with the other two NPs, exhibited a Corresponding experiments showed that the LENK peptide was stronger effect with a shorter duration, probably because the enzy- released from LENK-SQ-Diox and LENK-SQ-Dig but not from matic serum capability is not predictive of the enzymatic ecosystem LENK-SQ-Am (Fig. 3). In the case of LENK-SQ-Diox and LENK- in the inflamed paw. LENK-SQ-Dig and LENK-SQ-Diox NPs had SQ-Dig, the release of the respective LENK and LENK linker frag- nearly the same antihyperalgesic profile with prolonged effect, ment was followed by a progressive degradation of the peptide, due resulting in a significantly higher AUC than morphine and LENK- to serum enkephalinases (Fig. 3, A and B). In the case of the LENK- SQ-Am NPs. In particular, the LENK-SQ-Dig NPs showed an anti- SQ-Diox bioconjugate, both the LENK and the SQ moieties were hyperalgesic effect that lasted twice as long as morphine. In addition, each linked to the dioxycarbonyl linker through an ester bond. as expected for an analgesic compound, PWL values after morphine In the case of the LENK-SQ-Dig bioconjugate, the diglycolate linker -carrageenan–treated rats exceeded basal values in  treatment in was attached on one side to squalene by an ester bond and on the naïve healthy rats, whereas, in contrast, under the conditions other side to the LENK through an amide bond; in the LENK-SQ- used here, PWL values after LENK-SQ NPs just reached these Am bioconjugate, a direct amide bond connected the LENK to the basal values (Fig. 5). This would suggest that LENK NPs are devoid 5 of 12 : eaau5148 13 February 2019 5 2019; Sci. Adv. et al ., Feng

6 | SCIENCE ADVANCES RESEARCH ARTICLE Downloaded from http://advances.sciencemag.org/ on May 5, 2019 ), LENK- D Fig. 5. Antihyperalgesic effect of LENK-SQ NPs and morphine. Antihyperalgesic effects of acute treatment with morphine ( A and B ), LENK-SQ-Diox NPs ( C and H ) in E and F ), and LENK-SQ-Am NPs ( G and SQ-Dig NPs (  -carrageenan–induced inflammatory pain injected rats. Administration of morphine, LENK-SQ NPs, Nal, Nal-M, LENK, blank SQ NPs, or dextrose solution (vehicle) was performed (arrows, 0 on abscissa) 3 hours after  -carrageenan injection into the right hind paw. Morphine (A), LENK-SQ- SEM of independent determinations in five to nine animals Diox NPs (C), LENK-SQ-Dig NPs (E), and LENK-SQ-Am NPs (G) induced an increase in PWL (in seconds, means ± $$ ### $ < 0.001, compared to dextrose solution or LENK solution; per group) in the Hargreaves test. * < < 0.01, *** P 0.05, ** P P P < 0.001, compared to morp hine; P < 0.05, P < 0.01, $$$ P < 0.001, compared to LENK-SQ NPs. Two-way analysis of variance (ANOVA) with repeated measures, Bonferroni post test. Nal or Nal-M was administered 15 min before morphine or LENK-SQ NP injection. Basal on abscissa: Control (naïve) rats (before  -carrageenan injection). (B, D, F, and H) Bars are the means ± SEM of AUCs (seconds × minutes) of the cumulative durations derived from the time course changes (A, C, E, and G) in PWL after the various treatments. * < 0.05, ** P < 0.01, *** P < 0.001, one-way P $$$ $ $$ ANOVA, Tukey post test, compared to dextrose (vehicle) or LENK solution; 0.05, P P < < 0.01, P < 0.001, compared to LENK-SQ NPs. 6 of 12 : eaau5148 13 February 2019 5 2019; et al Sci. Adv. ., Feng

7 | RESEARCH ARTICLE SCIENCE ADVANCES Downloaded from http://advances.sciencemag.org/ on May 5, 2019 Fig. 6. IVIS Lumina scan of mice and of their organs after intravenous administration of fluorescent LENK-SQ-Am NPs or control fluorescent dye solution (ven- tral view). ( A ) Biodistribution of fluorescent LENK-SQ-Am NPs in mice with inflamed right hind paw. ( B ) Biodistribution of fluorescent LENK-SQ-Am NPs in mice with ) Biodistribution of free dye in mice with inflamed right paw. ( hours. non-inflamed hind paw (saline injected only into the right hind paw). ( C D ) Zoom of group A at 2 left hind paw. hours. ( ( ) Zoom of group B at 2 ) Quantitative analysis of the paws with the same region of interest (ROI). R, right hind paw; L, F E of analgesic properties but are especially potent to counteract hyper- using in vivo fluorescence imaging in a mouse carrageenan-induced algesia in subjects suffering from chronic pain. However, further paw edema model. Our data highlighted the ability of the NPs to studies are required to assess this hypothesis. gain access to the peripheral inflamed tissue. Intravenous injection Pretreatment with Nal (a prototypical opioid antagonist) prevented earing b of fluorescent DiD–labeled LENK-SQ NPs in inflammation- the increase in PWL evoked by LENK-SQ NPs and morphine, indi- - mice resulted in a marked increase of fluorescence within the in cating that the antihyperalgesic effect of all these compounds was flamed hind paw, up to a level that is threefold higher than that in mediated by opioid receptors. On the other hand, pretreatment with the contralateral non-inflamed paw or in the paw of mice treated )] only marginally decreased 24 Nal-M [which does not cross the BBB ( -carrageenan). The very low accumula- with saline only (instead of  the antihyperalgesic effect of morphine, suggesting that the drug - tion of fluorescence in the non-inflamed paw and in the brain con acted mainly through central and, to a lesser extent, peripheral opi- firmed that the antihyperalgesic effect resulted from the targeting oid receptors. In contrast, Nal-M pretreatment abolished the anti- of LENK-SQ NPs toward peripheral opioid receptors in inflamed hyperalgesic effect of LENK-SQ NPs, which demonstrated that all tissue rather than toward central opioid receptors. It is likely that three LENK-SQ NPs acted exclusively through peripherally located the LENK-SQ distributed within the inflamed area as NPs rather opioid receptors. than in a single LENK-SQ molecular form. Whole-body imaging in To investigate the ability of LENK-SQ NPs to address the peptide Fig. 6 shows strong fluorescence in the inflamed paw, which was toward the inflamed tissue, biodistribution studies were performed not the case when the fluorescent dye was injected as a free compound. et al Feng : eaau5148 13 February 2019 5 2019; Sci. Adv. ., 7 of 12

8 | SCIENCE ADVANCES RESEARCH ARTICLE In addition, after incubation of LENK-SQ NPs in serum, a significant pounds were routinely conducted in glassware, which was flame-dried proportion of NPs remained intact (fig. S8). Last, safety of LENK-SQ under a positive pressure of nitrogen. HPLC-grade acetonitrile NPs after intravenous injection was confirmed by normal levels of (ACN), MeOH, ethanol (EtOH), and ethyl acetate (AcOEt) were transaminases and normal histology of vital organs. provided by Carlo Erba (Rodano, Italy). Thus, the novelty of the approach resulted from the unexpected ability of the peptidic NPs to target the small area of the body where Synthesis of LENK-SQ-Diox inflammation and nociception occur. As this resulted in an effective 1,1 ′ ,2-Tris-norsqualenic acid chloromethyl ester pain alleviation, which lasted even longer than with morphine and 1,1 ′ ,2-Tris-norsqualenic acid was synthesized by oxidation of 1,1 ′ ,2- avoided any diffusion into the CNS, such properties of LENK-SQ tris- norsqualenic aldehyde by Jones reagent as previously reported (300 mg, 3.0 mmol) in water (2 ml) NPs might open novel perspectives for pain management. 35 , ( 36 ). A solution of KHCO 3 was added to a solution of 1,1 ′ ,2-tris-norsqualenic acid (400 mg, NHSO (34 mg, 0.1 mmol) in DCM (2 ml). The 1 mmol) and n -Bu 4 4 CONCLUSION reaction mixture was vigorously stirred, and chloromethyl chloro- On the basis of the bioconjugation of LENK to squalene, we describe sulfate (185 mg, 1.15 mmol) was added dropwise. After stirring for here a new nanoformulation capable of precise and efficient delivery 1 hour, DCM (10 ml) was added to extract the product. The organic of LENK for pain control associated with inflammatory events. Our phase was separated, washed with brine, dried over magnesium sul- data demonstrated that the antihyperalgesic activity of LENK-SQ NPs fate, and concentrated under reduced pressure to afford a pale yellow took place at the level of peripheral opioid receptors. The experi- oil that was used in the following step without further purification. Downloaded from mental approach to making these nanoparticles is simple and easy Alloc-Leu-enkephalin-squalene (Alloc-LENK-SQ-Diox) (i.e., it does not require any complicated nanoparticle surface func- ,2-tris-norsqualenic acid chloromethyl ester (200 mg, ′ The 1,1 tionalization), which should facilitate further pharmaceutical devel- 0.445 mmol) was added into a mixture of Alloc-LENK (285 mg, (37 mg, 0.4 mmol) in 3 ml of DMF. The opment and clinical translation. Although further studies are needed to 0.445 mmol) and NaHCO 3 mixture was stirred at 40°C under argon for 4 days. The reaction more precisely determine how dosage, administration frequency, and final reaction mixture was concentrated in vacuo, and the residue timing of treatment with LENK-SQ may affect the clinical outcome, http://advances.sciencemag.org/ was purified by flash column chromatography on silica gel DCM/ this study opens a new exciting perspective for an efficient treatment EtOH (100:0 to 97:3) to afford the title compound as a pale yellow of intense pain, which evades the severe side effects associated with oil (168 mg, 40% yield). morphine or related synthetic opioids. Last, because of the versatility Leu-enkephalin-squalene with dioxycarbonyl of the approach, the application of this delivery system to other linker (LENK-SQ-Diox) therapeutic peptide molecules may be reasonably envisioned. TES (1215 mg, 10 mmol) was added dropwise neat to a stirred solu- tion of Alloc-LENK-SQ (110 mg, 0.1 mmol) and 10% Pd-C (20% by MATERIALS AND METHODS weight of Alloc-LENK-SQ-Diox) in MeOH (11 ml) under argon. Materials When the reaction was completed, the mixture was filtered through All the chemicals used were of analytical grade. Squalene (SQ), celite to remove the Pd-C, and the residual TES and solvent were on May 5, 2019 diglycolic anhydride, ethyl chloroformate, chloromethyl chloro- removed by evaporation. The residue was first purified by flash col- NHSO , TES, and triethylamine -Bu n sulfate, ammonium acetate, umn chromatography on silica gel with DCM/EtOH (90:10). The 4 4 (TEA) were purchased from Sigma-Aldrich (France). Pd-C was ob- resulting product was dissolved in 200  l of EtOH before undergo- tained from Alfa Aesar (France). DiD and DiR were purchased, ing a second purification via the semi-preparative RP-HPLC system respectively, from PromoKine (Germany) and Interchim (France). (Waters, MA, USA) on an Uptisphere C18 column (100 mm Roti-Histofix 4% (formaldehyde) was provided by Roth (Germany). by 21.2 mm; pore size, 5 m; Interchim, CA, USA) to obtain the  All the drugs used were of analytical grade. Morphine sulfate salt pure product (23 mg; 23% yield). HPLC was then performed using pentahydrate, ,   , and -carrageenan, naloxone hydrochloride (Nal,  a gradient elution with the mobile phase composed of an ammonium  opioid receptors antagonist), and naloxone methiodide (Nal-M, a acetate buffer (20 mM) and ACN. Elution was carried out at a flow nonspecific opioid receptor antagonist that does not cross the BBB) ml/min for 10 min with the linear gradient from 10 to rate of 21 were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). - 100% ACN, and then, the system was held at 100% ACN with iso Ketamine and xylazine were purchased from Centravet (Maisons- min. Temperature was set at 30°C, and ultraviolet cratic flow for 10 Alfort, France). LENK and Alloc-LENK were purchased from (UV) detection was monitored at 280 and 257 nm. The retention Ontores Biotechnologies (Zhejiang, China). time was 15 min, and the total yield of the pure product, after cou- pling and deprotection steps, corresponded to 9.5%. General information on chemicals Analytical thin-layer chromatography was performed on Merck silica Synthesis of LENK-SQ-Dig gel 60F254 glass precoated plates (0.25 mm layer). Column chromatog - ,2-Tris-norsqualenol was synthesized from squalene via 1,1 ′ 1,1 ,2- ′ raphy was performed on Merck silica gel 60 (230-400 mesh). HPLC tris-norsqualenic aldehyde according to previously reported methods water was purified using a Milli-Q system (Millipore, France). Tetra - , ( 35 36 ). Diglycolic anhydride (150 mg, 1.29 mmol) was added to a hydrofuran (THF) was distilled from sodium/benzophenone ketyl. solution of 1,1 ′ ,2-tris-norsqualenol (200 mg, 0.52 mmol) in 3 ml of - Dimethylformamide (DMF), dichloromethane (DCM), and pyri dry pyridine. The reaction was stirred overnight at room tempera- ) before distillation under dine were dried on calcium hydride (CaH ture. The solvent was removed and the residue was extracted with 2 an argon atmosphere. Methanol (MeOH) was dried over magnesium DCM from dilute hydrochloric acid and brine. Conversion to the and distilled. All reactions involving air- or water-sensitive com- squalene–diglycolic acid, monitored by TLC, was approximately 100%. 5 : eaau5148 13 February 2019 8 of 12 et al Sci. Adv. ., 2019; Feng

9 | RESEARCH ARTICLE SCIENCE ADVANCES The resultant product was dried under vacuum and used in the follow- or in 0.1 mM KCl (zeta potential). The results represent the mean ing step without further purification. Ethyl chloroformate (10.8 mg, and SD of three repeated sample preparations or more. 0.1 mmol) was added to a solution of squalene–diglycolic acid Cryo-TEM (50 mg, 0.1 mmol) and TEA (12 mg, 0.12 mmol) in 1 ml of anhy- The morphology of the LENK-SQ NPs was investigated by cryo-TEM. drous THF under argon at 0°C. The reaction was stirred for 1 hour NPs were vitrified using a chamber designed and set up in the labo- at room temperature, and a solution of LENK (55 mg, 0.1 mmol) in ratory where both humidity and temperature could be controlled. 1 ml anhydrous DMF was added. The mixture was maintained at Four microliters of solution of LENK-SQ NPs (4 mg/ml in Milli-Q 40°C for 2 days with stirring under argon. The solvents were removed water) was deposited onto a perforated carbon film mounted on a in vacuo, and the crude product was purified using silica gel chroma- 200-mesh electron microscopy grid. The homemade carbon film tography (purified with gradient eluent DCM/EtOH: 100:0 to 90:10). hole dimensions were about 2 mm in diameter. Most of the drop Then, ammonium salt was eliminated by simple filtration on silica was removed with a blotting filter paper, and the residual thin films using EtOH/AcOEt (40:60) as solvents. The pure bioconjugate was remaining within the holes were quick-frozen by plunging them . The specimen was then obtained with 69% yield. into liquid ethane cooled with liquid N 2 , to a cryo-specimen holder and observed transferred, using liquid N 2 using a JEOL FEG-2010 electron microscope. Micrographs were re- Synthesis of LENK-SQ-Am corded at 200 kV under low-dose conditions at a magnification of ′ 1,1 ,2-Tris-norsqualenic acid (100 mg, 0.25 mmol) and TEA (34.79 mg, ×40,000 on SO-163 Kodak films. Micrographs were digitized using 0.3 mmol) were dissolved in 1.5 ml of anhydrous THF under argon, a film scanner (Super Coolscan 8000 ED, Nikon), and analyses were and ethyl chloroformate (27 mg, 0.25 mmol) was added to the mix- Downloaded from made using the ImageJ software. ture at 0°C. The reaction was allowed to warm at room temperature and kept under stirring for 1 hour. A solution of LENK (138 mg, In vitro LENK release from NPs in serum 0.25 mmol) in 1.5 ml of anhydrous DMF was then added to the re- Frozen serum of male SWISS mice (900  l) was quickly thawed and action, and the mixture was kept under stirring for 2 days. The sol-  then preincubated at 37°C for 30 min before the addition of 300 l vents were removed in vacuo, and the crude product was purified of LENK-SQ-Dig NPs or LENK-SQ-Am NPs (2 mg/ml). In the case of twice using silica gel chromatography [purification with gradient http://advances.sciencemag.org/ LENK-SQ-Diox NPs, diluted serum (30% in 5% dextrose solution) eluent DCM/EtOH (100:0 to 90:10) and then simple filtration with was used for the release study. At various time intervals, aliquots EtOH/AcOEt (40:60) to remove the ammonium salt]. The pure bio-  (80  l of ACN to denature l) were collected and added into 320 conjugate was obtained with 73% yield. and precipitate the enzymes and proteins of the serum, to remove for 15 min). To quantify the residual g them after centrifugation (3000 Preparation and characterization of LENK-SQ NPs LENK-SQ bioconjugate and the released LENK, the resulting super- Preparation of nanoparticles natants (150  l) were evaporated to dryness at 40°C under nitrogen LENK-SQ NPs were prepared using the nanoprecipitation method- flow and then solubilized in 150  l of Milli-Q water. Free peptide ology. Briefly, the LENK-SQ bioconjugate (i.e., LENK-SQ-Diox, quantification was performed using RP-HPLC on an Uptisphere LENK-SQ-Dig, or LENK-SQ-Am) was dissolved in EtOH (8 mg/ml) Strategy C18HQ column (4.6 mm by 100 mm, 5 m; Interchim), a  and added dropwise under stirring (500 rpm) into a 5% aqueous on May 5, 2019 1525 Binary LC Pump (Waters), a 2707 Auto-sampler (Waters), dextrose solution (EtOH/dextrose solution volume ratio, 1:4). The and a 2998 PDA detector (Waters). The HPLC was carried out using solution became spontaneously turbid with a Tyndall effect, indi- a gradient elution with the mobile phase composed of 5 mM ammo- cating the formation of the nanoparticles. EtOH was then completely nium acetate in Milli-Q water (phase A) and 5 mM ammonium ac- evaporated using a Rotavapor (80 rpm, 30°C, 30 mbar) to obtain an etate in ACN (phase B). Elution was carried out at a flow rate of aqueous suspension of pure LENK-SQ NPs (final concentration, 1 ml/min for 13 min with the linear gradient from 10 to 100% of B; 2 mg/ml). Blank SQ NPs (LENK-free NPs) were prepared by the then, the system was held at 100% of B with isocratic flow for 10 min. same method as described above by adding dropwise an ethanolic Temperature was set at 35°C, and UV detection was monitored at solution of squalenic acid into 5% aqueous dextrose solution. Fluo-  257 nm. The detection limit of the HPLC technique was 0.39 g/ml rescently labeled LENK-SQ NPs were also obtained by the same 2 = 0.99998) over for the peptide. This method exhibited linearity ( R procedure, except that the fluorescent probe DiD was solubilized in  the assayed concentration range (0.39 to 200 g/ml) and demon- the ethanolic phase together with the LENK-SQ-Am bioconjugate strated good precision with relative SD being all less than 2.01%. (DiD/LENK-SQ-Am ratio was 4 wt %) before addition to the dex- The accuracy corresponded to 96 ± 5%. trose solution. Fluorescence quencher LENK-SQ NPs were also prepared by the same way using DiR as a fluorescent probe (DiD/ General information on in vivo study DiR/LENK-SQ-Am ratio was 2:2:100 wt). The peptide drug loadings Animals into the NPs were expressed as percentage (%), calculated from the Adult male Sprague-Dawley rats (200 to 220 g on arrival, 280 to ratio between LENK peptide Mw and LENK-SQ bioconjugate Mw. 300 g at the time of experiments) and adult male Swiss mice (18 to The LENK-SQ NPs were regularly observed by cryo-TEM. All the 20 g on arrival, 22 to 25 g at the time of experiments) were purchased NPs were freshly prepared and used within 2 hours (conservation at from JANVIER LABS (France) for algesimetry tests and biodis- 4°C) before in vivo experiments. tribution, respectively. They were housed in a standard controlled DLS measurements environment (22° ± 1°C, 60% relative humidity, 12-hour light/12-hour The mean particle size, polydispersity index (PDI), and zeta poten- dark cycle, lights on at 8:00 a.m.) with food and water available ad tial were primarily evaluated by DLS (Nano ZS, Malvern; 173° libitum, without any handling for at least 1 week before being used for scattering angle at 25°C). The measurements were performed in experiments. In all cases, experiments were performed in conformity triplicate following appropriate dilution of the NPs in water (DLS) : eaau5148 13 February 2019 5 2019; Sci. Adv. ., et al 9 of 12 Feng

10 | SCIENCE ADVANCES RESEARCH ARTICLE with the guidelines of the Committee for Research and Ethical Issues NPs (20 mg/kg, equivalent to 11.48 mg/kg of LENK) or control un- 37 ) and ap- of the International Association for the Study of Pain ( conjugated SQ NPs (8.28 mg/kg) was used based on the maximal proved by the Animal Care Committee of the University Paris- Sud volume of LENK-SQ NPs that could be injected. in accordance with the principles of laboratory animal care and the European legislation 2010/63/EU. All efforts were made to reduce Biodistribution study in mice animal numbers and minimize their suffering, as defined in the spe- In vivo imaging biodistribution studies were performed after intra- cific agreement (registered under no. 7493-2016102520355414). l, 2 mg/ml venous injection of fluorescent LENK-SQ-Am NPs (250  containing 4% DiD) or control fluorescent DiD solution (250  l, Carrageenan-induced paw edema model g/ml in 5% dextrose solution) in shaved mice bearing -carrageenan–  80  Since the demonstration that indomethacin reduces inflammation induced inflammation. In parallel, control non-inflamed shaved -carrageenan, this acute model  caused by intraplantar injection of l of saline into the right hind paw instead of  mice (injected with 20 is widely accepted for screening compounds with anti-inflammatory -carrageenan) also received injection of fluorescent LENK-SQ NPs.  ). potentialities ( 38  -Carrageenan was dissolved in physiological The biodistribution of the NPs was recorded at 0.5, 2, 4, 6, and saline (NaCl, 0.9%) just before injection. Rats or mice received a 24 hours with the IVIS Lumina LT Series III system (Caliper Life  -carrageenan solution in the plantar single intraplantar injection of Sciences) using 640 nm excitation and 695 to 775 nm emission filters, 39 ) to induce inflammation. The region of the right hind paw ( 23 , respectively. During imaging, the mice were kept on the imaging -carrageenan dose corresponded to 100 l (2% solution,   injected stage under anesthesia with 2% isoflurane gas in oxygen flow (1 liter/min) w/v) for rat and 20  l (3% solution, w/v) for mice. Inflammation and were imaged in ventral position. Images and measures of fluo- Downloaded from  reached its maximum 3 hours after -carrageenan injection. Thermal rescence signals were acquired and analyzed with Living Imaging. nociceptive test was then performed on the ipsilateral inflamed To measure photon radiance, regions of interest (ROIs; threshold of hind paw. 35%) were selected on the paw of the mice, and average radiant ef- ficiency values were used for quantification. Threshold of ROI for Nociceptive behavioral study in rats the inflamed paw was then pasted on the non-inflamed paw to com- Thermal nociceptive test pare the radiance with the same region. http://advances.sciencemag.org/ Hypersensitivity to thermal nociceptive stimuli was assessed using In a separate experiment, fluorescent LENK-SQ NP–injected mice the Hargreaves test ( 23 ). Rats were placed individually in an open were deeply anesthetized with a mixture of ketamine (100 mg/kg, Plexiglas cylindrical chamber (20 cm in diameter, 35 cm high) on a i.p.) and xylazine (10 mg/kg, i.p.) before euthanasia by transcardiac 3-mm-thick transparent glass floor and allowed to habituate for at perfusion of 40 ml of saline (8 ml/min), until the fluid exiting the least 20 min before testing. A movable radiant heat source (Model right atrium was entirely clear. Then, liver, spleen, kidneys, heart, 7370; Ugo Basile Plantar Test, Italy) was positioned under the glass lungs, brain, and inflamed right hind paw were excised and imme- floor directly beneath the plantar surface of the right hind paw, and diately imaged with the imager. The fluorescence emitted was quan- the time (in seconds) that elapsed from switching on the radiant tified with Living Image software over the ROI (threshold of 20%). heat until paw withdrawal was measured automatically. A cutoff time of 20 s was established to prevent tissue damage. Each trial was Toxicity study in rats on May 5, 2019 repeated three times with 5-min intervals for basal threshold and Adult male Sprague-Dawley rats were injected with either LENK- two times spaced (2-min intervals) after NP treatments 3 hours after = 3 animals per group). n SQ-Am NPs (20 mg/kg) or 5% dextrose (  -carrageenan injection. The average of PWLs was calculated and At 24 or 48 hours after injection, the animals were anesthetized with expressed as means ± SEM. 2% isoflurane gas in oxygen flow (1 liter/min), and blood was col- Experimental design for algesimetry test lected from tail blood vessels by anticoagulant (heparin, 500 UI/ml)– Basal responses to thermal stimuli were obtained on the day before treated syringes. The blood samples were centrifuged at 1000 for g  -carrageenan injection. On the basis of previous studies, acute the 15 min, and the plasma was collected and stored at −20°C before pharmacological treatments were performed 3 hours after carra- analysis. The AST and ALT levels in plasma were analyzed by Cerba geenan injection, which corresponded to the peak inflammatory Vet, France. response. The efficacy of these treatments on thermal hyperalgesia In a separate experiment, 24 or 48 hours after injection, rats were was evaluated by measurement of PWLs using the Hargreaves test deeply anesthetized by Dolethal. Liver, kidneys, spleen, heart, and at regular time intervals after drug or vehicle administration, first at lungs were then excised, fixed by 4% formaldehyde, paraffin-embedded, 10 min and then each at 30 min for a period of 4 hours (Fig. 4). m-thick sections. Hematoxylin and eosin staining was and cut into 5-  Acute pharmacological treatments performed on all the organs for analysis of the morphology (Zeiss). Morphine and LENK were dissolved in 5% dextrose solution, whereas Nal and Nal-M were dissolved in physiological saline (NaCl, 0.9%). All Statistical analyses - these drugs and NP suspensions were prepared just before administra All values are expressed as means ± SEM, and statistical analyses  -carrageenan tion. All acute treatments were performed 3 hours after were made with GraphPad Prism 6 software (San Diego, CA, USA). intraplantar injection according to Fig. 4. Nal and Nal-M were in- A two-way analysis of variance (ANOVA) was used with or without jected subcutaneously, whereas the intravenous route in the tail vein repeated measures as appropriate (see Materials and Methods and - was used for LENK-SQ NPs, LENK, and their controls. The antago legends to figures). The comparison between groups was performed nist (Nal or Nal-M) was administered 15 min before the agonist using the Bonferroni post hoc test. A one-way ANOVA followed by (morphine or tested NPs). A single dose of morphine (1 mg/kg), a Tukey post test was used to compare three or more groups in Nal (0.5 mg/kg), and Nal-M (0.5 mg/kg) was administered on the AUC bars. AUCs were calculated using the trapezoidal rule. For all ). A single intravenous dose of LENK-SQ 40 basis of literature data ( analyses, statistical significance was set at P ≤ 0.05. et al 10 of 12 : eaau5148 13 February 2019 5 Feng Sci. Adv. ., 2019;

11 | SCIENCE ADVANCES RESEARCH ARTICLE Y. Benson, Effect of formulation factors on incorporation of the E. Chen, F. 21. Wang, H. A. SUPPLEMENTARY MATERIALS Biopolymers 90 , hydrophilic peptide dalargin into PLGA and mPEG-PLGA nanoparticles. Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ 644–650 (2008). content/full/5/2/eaau5148/DC1 22. V. S. Chernyshev, R. Rachamadugu, Y. H. Tseng, D. M. Belnap, Y. Jia, K. J. Branch, Supplementary Text Skliar, Size and shape characterization of E. Butterfield, L. F. Pease III, P. S. Bernard, M. A. Fig. S1. Synthesis of LENK-SQ-Diox. hydrated and desiccated exosomes. Anal. Bioanal. Chem. 407 , 3285–3301 (2015). Fig. S2. Synthesis of LENK-SQ-Dig. Joris, A new and sensitive method for Brown, C. Dubner, F. Hargreaves, R. 23. K. Flores, J. Fig. S3. Synthesis of LENK-SQ-Am. 1 32 , 77–88 (1988). measuring thermal nociception in cutaneous hyperalgesia. Pain H spectrum of LENK-SQ bioconjugates. 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Couvreur, A unique squalenoylated and Bourgaux, B. Lepeltier, C. E. Frébourg (Electron Microscopy Facility/FR 3631-CNRS-UPMC) Châtenay-Malabry, France) and G. nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and Varna-Pannerec (Institut We thank M. are acknowledged for their contribution to cryo-TEM. anticancer activity. Proc. Natl. Acad. Sci. U.S.A. 111 , E217–E226 (2014). Galien Paris-Sud, Châtenay-Malabry, France) and D. Courilleau (CIBLOT Plateforme, P. 19. K. Mandal, J. S. McMurray, Pd−C-induced catalytic transfer hydrogenation with Dejean (BioCIS, Châtenay-Malabry, France) for help concerning the histological study. C. , 6599–6601 (2007). 72 Org. Chem. J. triethylsilane. Châtenay-Malabry, France) is acknowledged for help with the nuclear magnetic resonance 20. B. V. Banga, Liposomal formulation and characterization of K. Betageri, N. Vutla, A. G. J.F. is a fellow of the Chinese Scholarship Council (CSC). Part of this interpretations. Funding: 3 the opioid peptide leucine enkephalin. Pharm. Pharmacol. Commun. , 587–591 work was supported by the RBUCE-UP grant agreement (no. 00001002483/78) between the (2011). ERC and Université Paris-Sud, by the ERC under the Framework Program FP7/2007-2013 (grant 2019; 5 : eaau5148 13 February 2019 11 of 12 et al Sci. Adv. ., Feng

12 | RESEARCH ARTICLE SCIENCE ADVANCES agreement no. 249835), and by the Centre National de la Recherche Scientifique. UMR 8612 (no. 18306002.9, filed 23 July 2018). The other authors declare that they have no competing (P.C. team) is a member of the laboratory of excellence NANOSACLAY. Author contributions: interests. Data and materials availability: All data needed to evaluate the conclusions in P.C. and S.L.-M. were involved in planning and supervised the work. J.F. and S.L.-M. conceived the paper are present in the paper and/or the Supplementary Materials. Additional data and designed the bioconjugates. P.C. and S.L.-M. conceived and planned the experiments. related to this paper may be requested from the authors. J.F., S.L.-M., and P.C. developed the methodology. M.H. and A.G. developed the experimental design for algesimetry and F.C. attended meetings. J.F. carried out the experiments. Submitted 19 June 2018 A.G. participated in algesimetry experiments, and S.M. and C.C. participated in biodistribution Accepted 28 December 2018 studies. P.C., J.F., and S.L.-M. contributed to the analysis and interpretation of the results. Published 13 February 2019 M.H. contributed to the interpretation of the results of the behavioral study. J.F. wrote the 10.1126/sciadv.aau5148 manuscript (in consultation with S.L.-M. and P.C.). S.L.-M. and P.C. revised the manuscript. M.H. and A.G. revised the nociceptive behavioral study part. S.M. and C.C. participated in the Mura, C. Coudore, M. Hamon, Gautier, S. Cailleau, F. Lepetre-Mouelhi, A. Feng, S. J. Citation: P.C., J.F., and S.L.-M. are inventors on a European Competing interests: manuscript revision. Couvreur, A new painkiller nanomedicine to bypass the blood-brain barrier and the use of P. Application patent related to this work filed by CNRS and the University of Paris-Sud , eaau5148 (2019). morphine. Sci. Adv. 5 Downloaded from http://advances.sciencemag.org/ on May 5, 2019 et al 12 of 12 : eaau5148 13 February 2019 5 2019; Sci. Adv. ., Feng

13 A new painkiller nanomedicine to bypass the blood-brain barrier and the use of morphine Jiao Feng, Sinda Lepetre-Mouelhi, Anne Gautier, Simona Mura, Catherine Cailleau, François Coudore, Michel Hamon and Patrick Couvreur (2), eaau5148. 5 Sci Adv DOI: 10.1126/sciadv.aau5148 Downloaded from ARTICLE TOOLS http://advances.sciencemag.org/content/5/2/eaau5148 SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2019/02/11/5.2.eaau5148.DC1 MATERIALS http://advances.sciencemag.org/ REFERENCES This article cites 40 articles, 4 of which you can access for free http://advances.sciencemag.org/content/5/2/eaau5148#BIBL PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions on May 5, 2019 Use of this article is subject to the Terms of Service (ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 New Science Advances York Avenue NW, Washington, DC 20005. 2017 © The Authors, some rights reserved; exclusive licensee American Science Advances is a Association for the Advancement of Science. No claim to original U.S. Government Works. The title registered trademark of AAAS.

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