Article pubs.acs.org/molecularpharmaceutics
Stealth Liposomes Encapsulating Zoledronic Acid: A New Opportunity To Treat Neuropathic Pain Michele Caraglia,† Livio Luongo,‡ Giuseppina Salzano,§ Silvia Zappavigna,† Monica Marra,† Francesca Guida,§ Sara Lusa,§ Catia Giordano,‡ Vito De Novellis,‡ Francesco Rossi,‡ Alberto Abbruzzese Saccardi,† Giuseppe De Rosa,*,§ and Sabatino Maione‡ †
Department of Biochemistry and Biophysics “F. Cedrangolo” and ‡Department of Experimental Medicine, Second University of Naples, Via Costantinopoli, 16 80138 Naples, Italy § Department of Pharmacy, University Federico II of Naples, Via Montesano, 49, 80131 Naples, Italy ABSTRACT: In the pathogenesis of neuropathic pain, the conversion of astrocytes in the reactive state and the rasdependent Erk-mediated pathway play an important role. Zoledronic acid (ZOL) is a potent inhibitor of the latter pathway, but its activity in neurological diseases is hampered by its biodistribution that is almost exclusively limited to the bone. We have developed nanotechnological devices able to increase the accumulation of ZOL in extra bone sites. In this work, we have evaluated the effects of ZOL-encapsulating PEGylated liposomes (LipoZOL) on an animal model of neuropathic pain. We have found that 2 iv administrations (10 μg of ZOL, either as free or encapsulated into liposomes) at days 2 and 4 after the injury markedly reduced mechanical hypersensitivity at 3 and 7 days after nerve injury. On the other hand, free ZOL did not exert any significant alteration of the mechanical threshold. Immunohistochemical analysis of spinal cord revealed that GFAP-labeled astrocytes appeared hypertrophic activated cells in the ispilateral dorsal horn of spinal cord 7 days after SNI. LipoZOL significantly changed astrocyte morphology, by inducing a protective phenotype, without changing the total cell number. Moreover, the astrocytes of the spinal cord of LipoZOL-treated mice were positive for interleukin-10. Delivery of ZOL into the CNS was confirmed by biodistribution of fluorescently labeled liposomes. In particular, liposomes accumulated in the liver and kidney in both groups of normal and neuropathic animals; on the other hand, only in the case of neuropathic animals, a fluorescence increase in the brain and spinal cord occurred only in neuropathic animals at 30 min and 1 h. These data demonstrate that ZOL, only by using a delivery system able to cross the altered BBB, could be a new opportunity to treat neuropathic pain. KEYWORDS: stealth liposomes, zoledronic acid, aminobisphosphonates, neuropathic pain, liposome biodistribution
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proliferating status.5 After external damage leading to a pathological condition, they can pass toward a reactive status, participating in the processes leading to the occurrence of neurological diseases.6−8 In fact, glial and microglial cells are involved in the neuronal sensitization occurring in the dorsal horn of the spinal cord after peripheral nerve injury-induced neuropathic pain.9−11 In detail, microglia and then astrocytes are activated by the fiber damage and by the neuronal release of several nociceptive mediators such as ATP, glutamate, and substance P (SP), thus inducing a spinal wind up phenomenon responsible for the establishment of mechanical allodynia, which represents the neurological symptom of chronic neuropathic pain. Once
INTRODUCTION
Clinical management of chronic pain after nerve injury (neuropathic pain) and tumor invasion (cancer pain) is an objective difficult to achieve because the cell mechanisms that trigger and sustain chronic pain are not still definitively characterized. Glial cells, such as microglia and astrocytes in the central nervous system (CNS), are suggested to play an important role in the development and maintenance of chronic pain. In detail, chronic pain results from the occurrence of neural plasticity in both peripheral nervous system (PNS) and CNS.1−3 Glial cells are more abundant than neurons in the CNS and can be divided in three different groups: (i) astrocytes, (ii) microglia, and (iii) oligodendrocytes. Microglia are cells that display phenotypical signatures similar to macrophages, thus playing a scavenger function in CNS. The most present cell components for both number (about the half of glial cells) and size are astrocytes.4 Both microglia and astrocytes, in physiological conditions, are relatively not in a © 2013 American Chemical Society
Received: Revised: Accepted: Published: 1111
October 29, 2012 December 27, 2012 January 17, 2013 January 17, 2013 dx.doi.org/10.1021/mp3006215 | Mol. Pharmaceutics 2013, 10, 1111−1118
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achieving a terminal half-life of approximately 240 days.26 On this background, we designed a formulation based on stealth liposome-encapsulating ZOL (LipoZOL) to reduce the binding of ZOL to bone and to increase its bioavailability in extraskeletal tissue. We have demonstrated that, with this new liposome-based formulation, ZOL had increased antitumor properties as compared to standard (free) ZOL, using both in vitro and in vivo models of different human cancers.27 It has been well demonstrated that stealth liposomes can be used to deliver drugs into the CNS in pathological states in which BBB permeability is altered.28 In the case of chronic neuropathic pain, BBB is partially or totally disrupted and could allow the passage of nanovectors such as LipoZOL. In light of these considerations, we have investigated the antinociceptive effect of ZOL, administered as free or as LipoZOL, in an animal model of neuropathic pain. Moreover, levels of proinflammatory cytokines and CNS accumulation of LipoZOL were evaluated.
activated, glial cells are able to release pro-inflammatory cytokines and chemokines such as CCL2, IL-1β, and TNFα, thus continuously sensitizing neurons by interacting with their specific receptors. The pro-inflammatory state of these cells is also associated with a change in morphology that becomes hypertrophic and/or round shape, retracting the processes.12 In the last process, astrocytes overexpress intermediate filament proteins, such as vimentin, nestin, and glial fibrillary acidic protein (GFAP), proteoglycans, and other molecules that induce axon growth inhibition. The most important change found in astrocytes is not induction of proliferation but cytoplasm enlargement and migration toward the sites where damage was generated. Epidermal growth factor (EGF) and transforming growth factor α (TGFα) are likely involved in the wake up of astrocytes as it has been described that EGF receptor can be upregulated and hyperactivated in astrocytes after damage to the CNS.13 However, the downstream signal transduction components activated by EGF in these conditions have not been completely defined. Recent findings suggest an important role played by mitogenactivated protein kinases (MAPKs)ERK, p38, and JNKin the development of neuropathic pain.14 Interestingly, the three different MAPKs have a different timing of activation in spinal cord glial cells after nerve damage. In fact, p38 kinase is persistently stimulated, ERK is activated only in the early stages,15−17 while pERK induction has a late onset (more than 21 days from the damage).18,19 The intracranial administration of a MEK inhibitor antagonizes both the late onset stimulation of ERK and the occurrence of the associated mechanical allodynia, suggesting a role for astrocytic ERK in sustaining chronic pain.19 ERK-1/2 and their cognate kinases can be under the control of the ras family proteins that usually trigger the MAPK cascades.20 Moreover, it was recently reported that the Rheb (a ras family member)−mTOR pathway is up-regulated in reactive astrocytes of the injured spinal cord. Zoledronic acid (ZOL) is a member of the pharmacological agents named as aminobisphosphonates (NBPs) that are agents indicated for the treatment of bone demineralization caused by both osteoporotic conditions and tumor metastases. ZOL acts as a potent inhibitor of farnesyl pyrophosphate synthase and completely abolishes the synthesis of both farnesylpyrophosphate and geranylgeranylpirophosphate, inhibiting isoprenylation processes. Therefore, ZOL suppresses prenylation of all small GTPases, including Ras family proteins. 20 The prenylation process is needed for the compartmentalization of ras proteins at the inner side of the plasma membrane where they are activated by external signals.21,22 Unfortunately, one of the most important limitations of ZOL is its pharmacokinetic profile. In fact, pharmacokinetic studies have demonstrated that ZOL, following a standard intravenous infusion, is still detectable in the plasma only for 1−2 h before its accumulation in the bone.23,24 In these conditions, ZOL estimated distribution and elimination plasma half-lives are 15 (t1/2) and 105 min (t1/2β), respectively, with a peak plasma concentration after the end of infusion (Cmax) of approximately 1 μM.25 Biodistribution studies in rats and dogs with single or multiple intravenous doses of 14C-labeled ZOL have shown that ZOL is avidly uptaken by bone, where it accumulates, maintaining high levels over the time. Moreover, ZOL is continuously and slowly released in the plasma from the bone,
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MATERIALS AND METHODS Materials. Phosphatidylcholine from egg yolk (EPC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were a kind gift from Lipoid GmbH (Cam, Switzerland). 22-[N-[(7-Nitro-21,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol (NBD cholesterol) was obtained by Invitrogen (Paisley, United Kingdom). Tetrabutylammonium bromide (TBA), cholesterol (Chol), ammonium chloride, ammonium tiocyanate, potassium phosphate dibasic, sodium phosphate dibasic, iron(III) chloride anhydrous, and Sephadex G-150 were purchased from Sigma Chemical Co. (St. Louis, MO). Lactose was obtained from New Fa.Dem (Naples, Italy). Analytical grade diethyl ether, methanol, chloroform, and 30% ammonia solution, as well as HPLC grade acetonitrile, were obtained from Carlo Erba (Milan, Italy). ZOL was a kind gift from Novartis (Novartis, Basel, Switzerland). Liposome Preparation. The liposomes were prepared by a modified reverse-phase evaporation technique as previously described.27 Briefly, an organic solution consisting of EPC/ Chol/DSPE-PEG 2000 (1:0.32:0.30 weight ratio) in chloroform/methanol (2:1 volume ratio) was placed in a roundbottom flask under nitrogen atmosphere, and the solvent was removed under vacuum in a rotary evaporator. Three milliliters of diethyl ether was then added to the lipid film, and the resulting solution was sonicated (bath-type sonicator, Branson 3510, Danbury, United States) for 30 min in presence of 1 mL of ammonium chloride buffer at pH 9.5 containing 75 mM ZOL and 58 mM lactose. Glass beads (Sigma) were also added to the flask to facilitate the formation of an emulsion. Then, the organic solvent was removed under vacuum at 30 °C by a rotary evaporator (Laborota 4010 digital, Heidolph, Schwabach, Germany) in nitrogen atmosphere. Once a viscous gel was obtained, the vacuum was broken, and the gel was agitated on vortex for about 1 min. The resulting dispersion was placed again in the rotary evaporator for about 15 min under vacuum. The liposome suspension was repeatedly passed through polycarbonate membrane (Nucleopore Track Membrane 25 mm, Whatman, Brentford, United Kingdom) with 0.4 μm pore size under nitrogen by using a thermobarrel extruder system (Northern Lipids Inc., Vancouver, BC, Canada). Then, unencapsulated ZOL was removed by passing the suspension through Sephadex G-150 column where the liposomes were eluted in an aqueous solution containing 58 mM lactose. After 1112
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(50 mg/kg, ip). The sciatic nerve was exposed. The tibial and common peroneal nerves were tightly ligated with 5.0 silk thread, leaving the sural nerve spared. Sham mice were anaesthetized, and the sciatic nerve was exposed at the same level but not ligated. Mechanical allodynia was measured by using the Dynamic Plantar Aesthesiometer (Ugo Basile, Varese, Italy). Mice were allowed to move freely in one of the two compartments of the enclosure positioned on the metal grid surface. A mechanical stimulus was delivered to the plantar surface of the mouse hind paw by an automated steel filament exerting an increasing force of 3 g per second. Nociceptive responses for mechanical sensitivity were measured in grams. Baseline thresholds were determined 6 days before starting the treatments. Each mouse served as its own control, and the responses were measured both before and after surgical procedures. The observer was blind to the treatments. Immunohistochemistry. Under deep pentobarbital anesthesia, mice were transcardially perfused with saline solution followed by 4% paraformaldehyde (PFA) in 0.1% PBS. The lumbar spinal cord was dissected, postfixed for 4 h in 4% PFA, cryoprotected for 72 h in 20% sucrose in 0.1 M phosphate buffer, and frozen in O.C.T. embedding compound. Transverse sections (20 μm) were cut by using a cryostat and then mounted onto slides. Slides were incubated overnight with primary antibody solutions for the glial cell marker rabbit poly clonal anti-GFAP (1:1000; Dako Cytomation, Denmark) or IL10 (goat anti- IL10 Santa Cruz, United States). Following incubation, sections were washed and incubated for 3 h with secondary antibody solution (donkey antirabbit or donkey antigoat IgG-conjugated Alexa FluorTM 488 and 568; 1:500; Molecular Probes, United States). Slides were washed, coverslipped with Vectashield medium (Vector Laboratories, United States), and analyzed under a Leica fluorescence microscope. Cell Preparation from Solid Tissues for FACS. Tissues were excised for either 30 s or 1, 3, 6, or 24 h after injection and minced into 2−4 mm pieces using scissors or scalpel blade. Tissue pieces were trypsinized and incubated at 37 °C for 15 min. Cells were dispersed by gentle pipetting and filtered through a cell strainer to eliminate clumps and debris. The cell suspension was collected in a conical tube and centrifuged for 4−5 min (300−400g) at 4 °C, discarding the supernatant. The cell pellet was washed in PBS to remove excess enzyme solution and then resuspended in the same buffer to perform a FACS analysis (FACScan, Becton Dickinson). For each sample, 2 × 104 events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments. CellQuest software (Becton Dickinson) was used to calculate mean fluorescence intensities (MFIs). The MFIs were calculated by the formula (MFItreated/MFIcontrol), where MFItreated is the fluorescence intensity of cells treated with Liposomes and MFIcontrol is the fluorescence intensity of untreated and unstained cells.
preparation, the liposome suspension was quickly frozen in liquid nitrogen and lyophilized for 24 h. Blank liposomes were prepared similarly. For FACS analysis, liposomes containing NBD cholesterol in a 1.7% weight ratio with respect to total Chol were prepared. All liposome preparations were stored at −20 °C. Each formulation was prepared in triplicate. Liposome Size. The liposome mean diameter and size distribution were measured by photon correlation spectroscopy (PCS) (N5, Beckman Coulter, Miami, United States) at 20 °C. Briefly, liposomes were diluted in deionizer/filtered water, and the measures were carried out with the detector set at a 90° angle. The particle size distribution was expressed as polydispersity index (PI). For each batch, the results were the mean of three measures. For each formulation, the mean diameter (reported in nm) and PI were calculated as the mean of three different batches. ZOL Encapsulation into Liposomes. ZOL loading into liposomes was expressed as ZOL actual loading and encapsulation efficiency. ZOL actual loading was calculated as μg of ZOL/mg of lipids in the freeze-dried powder; ZOL encapsulation efficiency was obtained as the ratio between ZOL loaded into liposomes (LipoZOL) and ZOL theoretical encapsulation. The phospholipid content of the liposome suspension was determined by the Stewart’s assay.29 Briefly, liposomes were added to an aqueous ammonium ferrithiocyanate solution (0.1 N) mixed with chloroform. The concentration of the phospholipids, namely, PC and DSPE-PEG, was calculated by measuring the absorbance at a wavelength of 485 nm into the organic layer. The quantitative analysis of ZOL was carried out by reverse-phase chromatography (RP-HPLC) on a Gemini 5 μm C18 column (250 mm × 4.60 mm, 110 Å, Phenomenex, Klwid, United States) coupled with a security guard. ZOL was eluted in isocratic conditions (flow rate, 1 mL/ min) with a mixture 20:80 (v/v) of acetonitrile/aqueous solution (8 mM dipotassium hydrogen orthophosphate, 2 mM disodium hydrogen orthophosphate, and 7 mM tetra-n-butyl ammonium hydrogen sulfate, adjusted to a pH of 7.0 with sodium hydroxide) at room temperature. The analysis was carried out with an isocratic pump (LC-10A VP, Shimadzu, Kyoto, Japan) equipped with a 7725i injection valve (Rheodyne, Cotati, United States), SPV-10A UV−Vis detector (Shimadzu) set at a wavelength of 220 nm. Acquisition and analyses of the chromatograms were carried out by a Class VP Client/Server 7.2.1 program (Shimadzu). ZOL dosage was carried out as follows. Briefly, 100 μL of liposome suspension was mixed with 400 μL of water and 500 μL of chloroform. After mixing on vortex, the emulsion was centrifuged at 55g for 10 min, and the surnatant was analyzed by RP-HPLC. Animals. Male CD1 mice (35−40 g) were positioned three per cage under room temperature (20−22 °C) and humidity (55−60%) and under controlled illumination (12:12 h light:dark cycle; light on 06.00 h) for at least 1 week before to begin the experimental procedures. Food and water were provided ad libitum. The experiments received the approval by the Animal Ethics Committee of the Second University of Naples. Animal care was carried out according to the IASP and European Community (E.C. L358/1 18/12/86) guidelines for the use and protection of animals in experimental research. Animal suffering and the number of mice used were reduced as possible. Spared Nerve Injury (SNI). Mononeuropathy was induced according to a previously described method of Decostered and Woolf.30 Mice were anaesthetized with sodium pentobarbital
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RESULTS Liposome Characteristics. Liposomes containing zoledronic acid (LipoZOL) had a mean diameter of about 241 ± 36. ZOL was successfully encapsulated into liposomes at an actual loading of 101.41 ± 38.4, with about 70% of ZOL still loaded into liposomes after freeze drying and following reconstitution in water, as previously reported.27 LipoZOL Reduced Thermal Hyperalgesia and Mechanical Allodynia in Mice with SNI. SNI was associated 1113
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hypersensitivity at 3 and 7 days after nerve injury (Figure 1). On the other hand, free ZOL (10 μg/dose) did not exert any significant alteration of the mechanical threshold (Figure 1). Immunohistochemistry. In the present study, we have evaluated the glial components associated with the establishment of spinal sensitization occurring in neuropathic pain. GFAP-labeled astrocytes appeared as hypertrophic-activated cells in the ispilateral dorsal horn of the spinal cord 7 days after SNI. Two LipoZOL administrations significantly changed the astrocyte morphology, by inducing a protective phenotype, without changing the total cell number (Figure 2). Moreover, double labeling revealed in the spinal cord of lipoZOL-treated mice positive profiles for IL-10, which was expressed by GFAPlabeled astrocytes (Figure 2). Liposome Biodistribution. To study the in vivo biodistribution of liposomes, we performed FACS analysis of different tissues collected at different times from mice injected with fluorescently labeled liposomes. Mice were randomized into two groups, neuropathic or not, and iv administered with a single dose of fluorescently labeled LipoZOL. In detail, we analyzed the LipoZOL content in cells from different solid tissues (liver, kidney, brain, spinal cord, lung, and ganglia) at 30 s and 1, 3, 6, and 24 h after injection. Unlabeled liposomes determined a not significant fluorescence that was absolutely overlapping that one induced by free ZOL. On this light, we have assumed unlabeled liposomes as negative control.
with the development of ipsilateral mechanical and thermal hypersensitivity that was assessed up to 7 days after peripheral nerve injury (Figure 1). Both contralateral and sham-operated
Figure 1. Effect of two intravenous treatments with 0.9% NaCl, white liposomes, ZOL, or lipozol (20 μg) on mechanical allodynia in sham and SNI mice. Data are reported as means ± SEMs from six to eight mice per group. * indicates a significant difference (P < 0.05) vs SNI/ veh. Data were analyzed by two-way ANOVA, followed by Student Neuman−Keuls’ posthoc test.
thresholds remained unaltered (data not shown). Two iv administrations (10 μg of ZOL encapsulated into liposomes) at days 2 and 4 after the injury markedly reduced mechanical
Figure 2. SNI induces an increase in the number of hypertrophyc astrocytes in the ipsilateral dorsal horn of the spinal cord as compared to the contralateral side (A, upper panel). LipoZOL treatment significantly reduces the number of hypertrophyc astrocytes in the ipsilateral side of the dorsal horn (A, lower panel). B represents LipoZOL-induced overexpression of IL-10 in the GFAP-labeled astrocytes. C represents quantitative analysis of GFAP-positive profiles. Data are expressed as the mean ± SEM. ANOVA Tukey test. Scale bar, 100 μm. 1114
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Figure 3. Right panels: NBD-cholesterol-labeled LipoZOL fluorescence determined in cells from liver (A), kidney (B), and lung (C) of neuropathic (SNI) and not neuropathic (CTR) animals expressed as a % of increase of the mean fluorescence intensity (MFI) as compared to that one induced by nonfluorescent liposomes (blank liposomes). FACS analysis of the LipoZOL content at 30 s and 1, 3, 6, and 24 h after injection of a single dose of LipoZOL. The MFIs were calculated, as described in the Materials and Methods. Values are the means of three independent experiments (±SD). Left panels: Representative histograms relative to fluorescence associated to NBD-cholesterol-labeled LipoZOL in cells derived from liver (A), kidney (B), and lung (C) of neuropathic (SNI) and not neuropathic (CTR) animals. Full blue histograms represent the fluorescence of the negative controls (nonfluorescent or blank liposomes).
of spinal cord, the increase of fluorescence associated with LipoZOL in neuropathic animals occurred at a later time point and at a lesser extent than that one recorded in brain: in fact, an about 190% increase of the fluorescence was recorded only at 1 h from the injection (Figure 4B). Similarly to the brain, the fluorescence in spinal cord of neuropathic animals decreased in a time-dependent manner becoming not significantly altered after 24 h from the injection (Figure 4B). Once again, fluorescence was not significantly increased in the spinal cord of normal mice (Figure 4B). The fluorescence associated with LipoZOL was evaluated also in ganglia of normal and neuropathic animals, and the results indicated no specific uptake of the liposomes in this specific tissue at both 6 and 24 h from the injection (Figure 4C). In the latter case, the fluorescence was evaluated only at two time points due to the poorness of the biological material associated with the ganglia of the mice. In conclusion, these results suggested a significant accumulation of the ZOL-containing liposomes in brains and spinal cords of neuropathic mice while their accumulation did not occur at all in the same tissues of normal mice.
As expected, FACS analysis revealed an increased LipoZOL uptake in liver and kidney of both mice groups, neuropathic or not. In detail, after 30 min, LipoZOL uptake was about 210 and 200% increased as compared to negative control in liver and kidney of both neuropathic and not neuropathic mice, respectively (Figure 3A,B). LipoZOL accumulation in the liver of both animal groups remained almost unchanged for all of the observation period, while liposome accumulation in kidney decreased in a time-dependent manner, reaching about 70−80% increase as compared to negative controls (Figure 3A,B). On the other hand, there were not significant changes in the LipoZOL content as compared to negative controls in the lungs of both animal groups (Figure 3C). The accumulation of the fluorescence associated to LipoZOL in the brain of normal animals was not significantly increased as compared to negative controls for all of the time of the observation. On the other hand, the increase of fluorescently labeled LipoZOL was about 100 and 150% increased in the brain of neuropathic mice at 30 min and 1 h after injection, respectively (Figure 4A). Thereafter, the fluorescence associated with LipoZOL decreased in a time-dependent manner reaching an about 35% increase at 24 h from the injection (Figure 4A). In the case 1115
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Figure 4. Right panels: NBD-cholesterol-labeled LipoZOL fluorescence determined in cells from brain (A), spinal cord (B), and ganglia (C) of neuropathic (SNI) and not neuropathic (CTR) animals expressed as a % of increase of the mean fluorescence intensity (MFI) as compared to that one induced by nonfluorescent liposomes (blank liposomes). FACS analysis of the LipoZOL content at 30 s and 1, 3, 6, and 24 h after injection of a single dose of LipoZOL. The MFIs were calculated, as described in the Materials and Methods. Values are the means of three independent experiments (±SD). Left panels: Representative histograms relative to fluorescence associated to NBD-cholesterol-labeled LipoZOL in cells derived from brain (A), spinal cord (B), and ganglia (C) of neuropathic (SNI) and not neuropathic (CTR) animals. Full blue histograms represent the fluorescence of the negative controls (nonfluorescent or blank liposomes).
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DISCUSSION
In healthy organisms, long circulating liposomes are not able to across the BBB.33 On the other hand, stealth nanovectors, such as PEGylated liposomes, can be efficiently used to deliver drug into the CNS, in the case of diseases characterized by an altered BBB.29 This has been shown in the case of an experimental model of tumor,34 multiple sclerosis,35,36 brain ischemia,37 and metastases.38 Taking into account the alterations of BBB found in experimental models of neuropathic pain,39 we hypothesized that stealth liposomes could allow the delivery of ZOL into CNS of SNI animal, to use ZOL as a modulator of neuropathic pain. FACS analysis has been recently proposed by different authors to follow the in vivo biodistribution of drug delivery systems, when radiolabeled drugs are not available.40−42 Intriguingly, through FACS analysis approach, we found significant fluorescence associated with liposomes encapsulating ZOL in the spinal cord in the case of SNI animals. On the other hand, fluorescence was not found in the case of healthy animals. Therefore, the second step was to confirm the hypothesis of ZOL delivery into the CNS by measuring the effect of LipoZOL on the mechanical allodynia. We found a significant reduction of mechanical hypersensitivity at 3 and 7 days after
In this work, a new approach for the treatment of the neuropathic pain was proposed and investigated. In particular, our hypothesis was that ZOL, an aminobisphosphonate used in the clinical setting to prevent skeletal-related events in bone metastasis, osteoporosis, or Paget’s disease, could be a new and powerful pharmacological agent to control neuropathic pain. However, following iv administration, ZOL rapidly accumulates into the bone (about 55% of the administered dose), with very low concentrations in extraskeletal tissues.23−25 In our previous works, we demonstrated that the use of nanovectors allows the escape from bone accumulation, thus increasing the ZOL concentration in nonskeletal tissues, that is, in extrabone sites of different kinds of human tumors.27,31,32 In fact, we have previously demonstrated efficient inhibition of tumor growth in different experimental cancer models, that is, prostate cancer and multiple myeloma, only when using stealth nanovectors encapsulating ZOL, while this effect was negligible with free ZOL. These data supported our hypothesis that the use of stealth nanovectors can change ZOL pharmacokinetics. 1116
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(7) Hanisch, U. K.; Kettenmann, H. Microglia: Active sensor and versatile effector cells in the normal and pathologic brain. Nat. Neurosci. 2007, 10, 1387−1394. (8) Scholz, J.; Woolf, C. J. The neuropathic pain triad: neurons, immune cells and glia. Nat. Neurosci. 2007, 10, 1361−1368. (9) Clark, A. K.; Yip, P. K.; Grist, J.; Gentry, C.; Staniland, A. A.; Marchand, F.; Dehvari, M.; Wotherspoon, G.; Winter, J.; Ullah, J.; Bevan, S.; Malcangio, M. Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 10655−10660. (10) Luongo, L.; Palazzo, E.; Tambaro, S.; Giordano, C.; Gatta, L.; Scafuro, M. A.; Rossi, F. S.; Lazzari, P.; Pani, L.; de Novellis, V.; Malcangio, M.; Maione, S. 1-(2′,4′-Dichlorophenyl)-6-methyl-Ncyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide, a novel CB2 agonist, alleviates neuropathic pain through functional microglial changes in mice. Neurobiol. Dis. 2010, 37, 177−185. (11) Luongo, L.; Sajic, M.; Grist, J.; Clark, A. K.; Maione, S.; Malcangio, M. Spinal changes associated with mechanical hypersensitivity in a model of Guillain-Barré syndrome. Neurosci. Lett. 2008, 437, 98−102. (12) Hains, B. C.; Waxman, S. G. Activated microglia contribute to the maintenance of chronic pain after spinal cord injury. J. Neurosci. 2006, 26, 4308−4317. (13) Liu, L.; Cash, T. P.; Jones, R. G.; Keith, B.; Thompson, C. B.; Simon, M. C. Hypoxia-induced energy stress regulates mRNA translation and cell growth. Mol. Cell 2006, 21, 521−531. (14) Ji, R. R.; Gereau, R. W.; Malcangio, M.; Strichartz, G. R. MAP kinase and pain. Brain Res. Rev. 2009, 60, 135−148. (15) Zhuang, Z. Y.; Wen, Y. R.; Zhang, D. R.; Borsello, T.; Bonny, C.; Strichartz, G. R.; Decosterd, I.; Ji, R. R. A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. J. Neurosci. 2006, 26, 3551−3560. (16) Jin, S. X.; Zhuang, Z. Y.; Woolf, C. J.; Ji, R. R. p38 mitogenactivated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J. Neurosci. 2003, 23, 4017− 4022. (17) Tsuda, M.; Mizokoshi, A.; Shigemoto-Mogami, Y.; Koizumi, S.; Inoue, K. Activation of p38 mitogen- activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia 2004, 45, 89−95. (18) Ma, W.; Quirion, R. Partial sciatic nerve ligation induces increase in the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus. Pain 2002, 99, 175−184. (19) Zhuang, Z. Y.; Gerner, P.; Woolf, C. J.; Ji, R. R. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 2005, 114, 149−159. (20) Caraglia, M.; Budillon, A.; Tagliaferri, P.; Marra, M.; Abbruzzese, A.; Caponigro, F. Isoprenylation of intracellular proteins as a new target for the therapy of human neoplasms: preclinical and clinical implications. Curr. Drug Targets 2005, 6, 301−23. (21) Hancock, J. F.; Magee, A. I.; Childs, J. E.; Marshall, C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 1989, 57, 1167−1177. (22) Aznar, S.; Lacal, J. C. Rho signals to cell growth and apoptosis. Cancer Lett. 2001, 165, 1−10. (23) Chen, T. Pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with bone metastases. J. Clin. Pharmacol. 2002, 42, 1228−1236. (24) Caraglia, M.; Santini, D.; Marra, M.; Vincenzi, B.; Tonini, G.; Budillon, A. Emerging anti-cancer molecular mechanisms of aminobisphosphonates. Endocr. Relat. Cancer 2006, 13, 7−26. (25) Caraglia, M.; Marra, M.; Naviglio, S.; Botti, G.; Addeo, R.; Abbruzzese, A. Zoledronic acid: An unending tale for an antiresorptive agent. Expert Opin. Pharmacother. 2010, 11, 141−154.
nerve injury, while no effect was found in the case of free ZOL, administered at the same dose, that is, 10 μg of ZOL at days 2 and 4 after the injury. Moreover, it has been recently reported that cortical plasticity is mandatory for maintaining tactile allodynia.43 Therefore, the balancing of the non-neuronal cell phenotypes in this pivotal area of the CNS is important for neuropathic pain management. Peripheral nerve injury is also associated with BBB alterations39 due to an overall inflammatory process that involves several cytotypes. Interestingly, in this study, the analgesic effect of LipoZOL occurred together with the restoration of normal glial architecture of the dorsal horn of the spinal cord, while free ZOL was not able to induce any restoring effect. These effects on the astrocyte shape toward a protective phenotype were not associated to changes in their total number. The phenotypical shift of astrocytes is also consistent with the increased expression of the antiinflammatory cytokine IL-10 induced by the LipoZOL treatment. In fact, in healthy SNI mice treated with vehicle as well as SNI animals treated with free ZOL, the IL-10 was undetectable immunohistochemically (not shown). Taken together, our results confirm that sciatic nerve damage-induced BBB alterations could promote the infiltration of the LipoZOL in the dorsal horn of spinal cord, thus leading to the right ZOL concentrations in the CNS able to modulate the phenotypical shift of glial cells and to abrogate neuropatic pain. Our study opens new perspectives for the treatment of neuropathic pain, providing a new and powerful approach based on the combined use of a potent aminobisphosphonate and stealth liposomes.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +39(0)81 678 666. Fax: +39(0)81 678 630. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS M.C. received a contribution from the Italian Ministry of Education and Research (MIUR, PRIN 2009), from the Italian Association for Cancer Research (AIRC) for a project entitled “Liposomes encapsulating zoledronic acid: a new experimental therapeutic for the treatment of brain tumors”, and from Regione Campania for “Laboratori Pubblici” Hauteville. This work is dedicated to the memory of the beloved Prof. Alberto Abbruzzese.
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REFERENCES
(1) Costigan, M.; Scholz, J.; Woolf, C. J. Neuropathic pain: A maladaptive response of the nervous system to damage. Annu. Rev. Neurosci. 2009, 32, 1−32. (2) Ji, R. R.; Kohno, T.; Moore, K. A.; Woolf, C. J. Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci. 2003, 26, 696−705. (3) Basbaum, A. I.; Bautista, D. M. G.; Scherrer; Julius, D. Cellular and molecular mechanisms of pain. Cell 2009, 139, 267−284. (4) Aldskogius, H.; Kozlova, E. N. Central neuron-glial and glial-glial interactions following axon injury. Prog. Neurobiol. 1998, 55, 1−26. (5) Nimmerjahn, A.; Kirchhoff, F.; Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 2005, 308, 1314−1318. (6) Rossi, D. J.; Brady, J. D.; Mohr, C. Astrocyte metabolism and signaling during brain ischemia. Nat. Neurosci. 2007, 10, 1377−1386. 1117
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(26) Weiss, H. M.; Pfaar, U.; Schweitzer, A.; Wiegand, H.; Skerjanec, A.; Schran, H. Biodistribution and plasma protein binding of zoledronic acid. Drug Metab. Dispos. 2008, 36, 2043−2049. (27) Marra, M.; Salzano, G.; Leonetti, C.; Tassone, P.; Scarsella, M.; Zappavigna, S.; Calimeri, T.; Franco, R.; Liguori, G.; Cigliana, G.; Ascani, R.; La Rotonda, M. I.; Abbruzzese Saccardi, A.; Tagliaferri, P.; Caraglia, M.; De Rosa, G. Nanotechnologies to use bisphosphonates as potent anticancer agents: the effects of zoledronic acid encapsulated into liposomes. Nanomedicine 2011, 7, 955−964. (28) De Rosa, G.; Salzano, G.; Caraglia, M.; Abbruzzese Saccardi, A. Nanotechnologies: a strategy to overcome blood-brain barrier. Curr. Drug Metab. 2012, 13, 61−69. (29) Stewart, J. C. M. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal. Biochem. 1980, 104, 10−14. (30) Decostered, I.; Woolf, C. J. Spared nerve injury: An animal model of persistent peripheral neuropathic pain. Pain 2000, 87 (2), 149−158. (31) Salzano, G.; Marra, M.; Porru, M.; Zappavigna, S.; Abbruzzese Saccardi, A.; La Rotonda, M. I.; Leonetti, C.; Caraglia, M.; De Rosa, G. Self-assembly nanoparticles for the delivery of bisphosphonates into tumors. Int. J. Pharm. 2011, 403, 292−297. (32) Marra, M.; Salzano, G.; Leonetti, C.; Porru, M.; Franco, R.; Zappavigna, S.; Liguori, G.; Botti, G.; Chieffi, P.; Lamberti, M.; Vitale, G.; Abbruzzese Saccardi, A.; La Rotonda, M. I.; De Rosa, G.; Caraglia, M. New self-assembly nanoparticles and stealth liposomes for the delivery of zoledronic acid: A comparative study. Biotechnol. Adv. 2012, 30, 302−309. (33) Huwyler, J.; Wu, D.; Pardridge, W. M. Brain drug delivery of small molecules using immunoliposomes. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14164−14169. (34) Siegal, T.; Horowitz, A.; Gabizon, A. Doxorubicin encapsulated in sterically stabilized liposomes for the treatment of a brain tumor model: biodistribution and therapeutic efficacy. J. Neurosurg. 1995, 83, 1029−1037. (35) Schmidt, J.; Metselaar, J. M.; Wauben, M. H.; Toyka, K. V.; Storm, G.; Gold, R. Drug targeting by long-circulating liposomal glucocorticosteroids increases therapeutic efficacy in a model of multiple sclerosis. Brain 2003, 126, 1895−1904. (36) Kizelsztein, P.; Ovadia, H.; Garbuzenko, O.; Sigal, A.; Barenholz, Y. Pegylated nanoliposomes remote-loaded with the antioxidant tempamine ameliorate experimental autoimmune encephalomyelitis. J. Neuroimmunol. 2009, 213, 20−25. (37) Fukumoto, D.; Kawaguchi, A. T.; Haida, M.; Yamano, M.; Ogata, Y.; Tsukada, H. Liposome-encapsulated hemoglobin reduces the size of cerebral infarction in rats: Effect of oxygen affinity. Artif. Organs 2009, 33, 159−163. (38) Caraglia, M.; Addeo, R.; Costanzo, R.; Montella, L.; Faiola, V.; Marra, M.; Abbruzzese Saccardi, A.; Palmieri, G.; Budillon, A.; Grillone, F.; Venuta, S.; Tagliaferri, P.; Del Prete, S. Phase II study of Temozolomide plus pegylated liposomal doxorubicin in the treatment of brain metastases from solid tumours. Cancer Chemother. Pharmacol. 2006, 57, 34−39. (39) Echeverry, S.; Shi, X. Q.; Rivest, S.; Zhang, J. Peripheral nerve injury alters blood-spinal cord barrier functional and molecular integrity through a selective inflammatory pathway. J. Neurosci. 2011, 31, 10819−10828. (40) Hagtvet, E.; Evjen, T. J.; Nilssen, E. A.; Olsen, D. R. Assessment of liposome biodistribution by non-invasive optical imaging: A feasibility study in tumour-bearing mice. J. Nanosci. Nanotechnol. 2012, 12, 2912−2918. (41) Hinton, T. M.; Monaghan, P.; Green, D.; Kooijmans, S. A.; Shi, S.; Breheney, K.; Tizard, M.; Nicolazzo, J. A.; Zelikin, A. N.; Wark, K. Biodistribution of polymer hydrogel capsules for the delivery of therapeutics. Acta Biomater. 2012, 8, 3251−3260. (42) Mérian, J.; Gravier, J.; Navarro, F.; Texier, I. Fluorescent nanoprobes dedicated to in vivo imaging: from preclinical validations to clinical translation. Molecules 2012, 17, 5564−5591. (43) Giordano, C.; Siniscalco, D.; Melisi, D.; Luongo, L.; Curcio, A.; Soukupova, M.; Palazzo, E.; Marabese, I.; De Chiaro, M.; Rimoli, M.
G.; Rossi, F.; Maione, S.; de Novellis, V. The galactosylation of N(ω)nitro-L-arginine enhances its anti-nocifensive or anti-allodynic effects by targeting glia in healthy and neuropathic mice. Eur. J. Pharmacol. 2011, 656, 52−62.
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