Apamin-Mediated Actively Targeted Drug Delivery for Treatment of

Article pubs.acs.org/molecularpharmaceutics

Apamin-Mediated Actively Targeted Drug Delivery for Treatment of Spinal Cord Injury: More Than Just a Concept Jin Wu,† Hong Jiang,† Qiuyan Bi,† Qingsong Luo,† Jianjun Li,† Yan Zhang,† Zhangbao Chen,† and Chong Li*,†,‡ †

College of Pharmaceutical Sciences, Southwest University, Chongqing, 400715, P. R. China Chongqing Engineering Research Center for Pharmaceutical Process and Quality Control, Chongqing, 400715, P. R. China

‡

S Supporting Information *

ABSTRACT: Faced with the complex medical challenge presented by spinal cord injuries (SCI) and considering the lack of any available curative therapy, the development of a novel method of delivering existing drugs or candidate agents can be perceived to be as important as the development of new therapeutic molecules. By combining three ingredients currently in clinical use or undergoing testing, we have designed a central nervous system targeted delivery system based on apaminmodified polymeric micelles (APM). Apamin, one of the major components of honey bee venom, serves as the targeting moiety, poly(ethylene glycol) (PEG) distearoylphosphatidylethanolamine (DSPE) serves as the drug-loaded material, and curcumin is used as the therapeutic agent. Apamin was conjugated with NHS (N-hydroxysuccinimide)-PEG-DSPE in a site-specific manner, and APM were prepared by a thin-film hydration method. A formulation comprising 0.5 mol % targeting ligand with 50 nm particle size showed strong targeting efficiency in vivo and was evaluated in pharmacodynamic assays. A 7-day treatment by daily intravenous administration of low doses of APM (corresponding to 5 mg/kg of curcumin) was performed. Significantly enhanced recovery and prolonged survival was found in the SCI mouse model, as compared to sham-treated groups, with no apparent toxicity. A single dose of apamin-conjugated polymers was about 700-fold lower than the LD50 amount, suggesting that APM and apamin have potential for clinical applications as spinal cord targeting ligand for delivery of agents in treatment of diseases of the central nervous system. KEYWORDS: apamin, curcumin, polymeric micelles, spinal cord injury, active targeted drug delivery the therapeutic efficacy of MP is relatively minor,4 while its side effects, including gastrointestinal bleeding, gastritis, and Cushing’s syndrome, are significant.5 Claims of improvements in functional outcomes after high doses of MP have recently become controversial, given the growing concern over the elevated risk of infection and higher mortality among spinal cord patients that received MP, particularly in view of its modest beneficial effects.6 Therefore, a wide variety of approaches have been explored to develop therapeutic alternatives, including macromolecules, natural products, stem cell mediated repair, and biomaterial engineering methods.7−9 As an illustrative example, multiple lines of evidence have validated the importance of the Rho pathway in controlling the neuronal responses to the growthinhibiting proteins following central nervous system (CNS) injury. Cethrin, a recombinant protein-based inhibitor of Rho,

1. INTRODUCTION Spinal cord injury (SCI), a pathological damage to the spinal cord resulting from trauma, inflammation, and many other causes,1 is a devastating condition that affects more than 130,000 people each year worldwide and often results in permanent functional and sensory deficits. The enormous impact, both in an individual and familial context and on the broader socioeconomical scale, is partly due to the early mean age (33 years) of the patient population, who are predominantly male (4:1 male/female ratio).2 Moreover, the lifelong supportive care needed to prevent complications (e.g., decubitus, respiratory and urinary tract infections, and others) means a substantial financial burden on both the patients and the society.2 Despite major progress in pharmacological and surgical treatment approaches, SCI remains a complex medical and psychological challenge, both for the patients and their relatives and for the involved physicians, with no curative therapy currently available. To date, methylprednisolone (MP) is the major recognized treatment for SCI, with neuroprotective effect that appear to be mediated through the inhibition of inflammatory reactions and lipid peroxidation.3 However, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3210

May 30, 2014 July 26, 2014 August 6, 2014 August 6, 2014 dx.doi.org/10.1021/mp500393m | Mol. Pharmaceutics 2014, 11, 3210−3222

Molecular Pharmaceutics

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altering the ratio of targeting ligand to the particle size. The therapeutic effects of APM on SCI were subsequently confirmed on mouse models by a variety of behavioral, histological, and electrophysiological methods. Additionally, a preliminary safety evaluation of potential toxicity of the peptide or peptide-modified polymers was performed.

has shown promise in treatment of traumatic SCI and is currently undergoing phase I/IIa clinical trials to evaluate its safety and tolerability.10,11 Among other examples, one of the most widely used biomedical polymers, polyethylene glycol (PEG), was found to be able to repair injured nerve cells after local subcutaneous administration by multiple distinct pathways, including resealing of the disrupted plasma membrane and the prevention of the effects of mitochondria-derived oxidative stress on the intracellular components.12,13 Additionally, natural products have elicited increasing interest, with a number of agents developed over the past decades showing effectiveness in the treatment of SCI, including curcumin, EGb 761 (extract of Ginkgo biloba), SM-216289 (isolated from the fermentation broth of a fungal strain), and many others.14−16 Their mechanisms of action were proposed to include the inhibition of nitric oxide synthesis, axonal regeneration, attenuation of neuronal loss, prevention of neuronal apoptosis, decreased astrocyte activation, and other regenerative responses which may benefit neuronal survival following SCI. Additionally, stem cells were reported to promote host neural repair in part by secreting growth factors, and their regenerationpromoting activities can be modified by gene delivery.17 The term “targeted therapy” refers to drug accumulation predominantly within the target zone, resulting in enhanced therapeutic efficacy and reduced drug concentration outside of the lesions, with increasing recognition of the importance of targeted drug delivery for therapeutic effectiveness.18−20 The active targeting approach, based on the specific ligand−receptor interactions between the drug carrier and the target cells, was proposed to improve target cell recognition and increase the uptake of delivered drug.21 Additionally, nanotechnology has played an important role in recent improvements in drug delivery through the remarkable properties arising from the nanosize effect, showing superiority over traditional formulations. For example, the enhanced permeability and retention (EPR) effect that arises from the encapsulation of the cytotoxic agent in 100 nm sized nanocarrier is reflected in significantly enhanced antitumor efficacy, helping bring several blockbuster drugs to the market.22 Furthermore, efficient drug accumulation in poorly permeable tumors and precise subcellular localization of drugs are achieved by the smaller drug delivery systems (30 nm).23,24 In comparison with numerous studies investigating optimal tumor-targeted drug delivery, research in the treatment of spinal cord related diseases through the development of novel drug delivery methods is relatively sparse.25−27 The aforementioned MP was administered primarily via intravenous injection, and Cethrin, a macromolecular drug, was used solely in local application. Therefore, combining the concept of targeted drug delivery and nanotechnology could be of great clinical significance and may offer promising future perspectives for the treatment of SCI. Based on the above information, a novel drug delivery system targeting the spinal cord for treating SCI was explored in this study using three ingredients currently in clinical use, or undergoing clinical testing. Apamin, a honey bee derived peptide with high penetration and specific distribution to the CNS, was used as a targeting ligand. Polyethyleneglycolcarbamyl distearoylphosphatidyl-ethanolamine (PEG-DSPE) micelles served as drug carriers, and curcumin (CUR) was chosen as model drug. Drug-loaded micelles were prepared and characterized through in vivo and in vitro studies. The current study systematically investigated the factors which may potentially influence drug targeting, including the effect of

2. MATERIALS AND METHODS 2.1. Materials and Animals. N-Hydroxysuccinimidyl (NHS)-PEG3400-DSPE was purchased from Nanocs Corp (New York, USA). mPEG2000-DSPE was supplied by AVT Pharmaceuticals, Ltd. (Shanghai, China). 1,1′-Dioctadecyl3,3,3,3′-tetramethylindotricarbocyanine iodide (DiR) was purchased from Invitrogen Corp. (Life Technologies, Carlsbad, CA, USA). Curcumin was obtained from Rongsheng ́ China). Other reagents were Biotechnology Co., Ltd. (Xian, all of analytical grade. Male Kunming mice (KM; 18−20 g) were obtained from the experimental animal center of Third Military Medical University (Chongqing, China) and kept under regulated conditions (12 h light/dark cycle, 21 °C) with free access to standard food and water. All animal experiments were performed in accordance with guidelines approved by the ethics committee of the College of Pharmaceutical Sciences, Southwest University. 2.2. Synthesis of Targeting Compound. Apamin and derivatives were chemically synthesized and folded as previously described.28 Peptides were then purified to homogeneity by reversed-phase high-performance liquid chromatography (HPLC), and their molecular weights were verified by electrospray ionization mass spectrometry (MS). The free amino group of the fourth lysine of apamin (CNCKAPETALCARRCQQH-NH2) was site-specifically conjugated with NHS-PEG3400-DSPE via Fmoc protection and deprotection in newly distilled N,N-dimethylformamide (DMF). After stirring for 24 h at room temperature, the reaction mixture was dialyzed against deionized water to remove the unreacted ligand.29,30 The solution was lyophilized and stored at −20 °C until required. The conjugation of apamin with PEG3400DSPE was confirmed by a matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF) MS (Autoflex speed, Bruker, Germany) using 7:3 acetonitrile:water with 0.1% trifluoroacetate as the matrix solution, with 10 mg/mL of α-cyano-4-hydroxycinnamic acid. 1H nuclear magnetic resonance (NMR) spectroscopy was used to assess the conjugation of apamin with PEG3400-DSPE. 2.3. Preparation and Characterization of Polymeric Micelles. 2.3.1. Preparation of Drug-Loaded Micelles. The CUR-loaded apamin-modified polymeric micelles (APM-CUR) were prepared by a thin-film hydration method as previously described.31 Briefly, apamin-PEG3400-DSPE and mPEG2000DSPE at 1:199 molar ratio were dissolved in methanol, and CUR was added in a 1:10 weight ratio (CUR:total polymers). Phosphate buffered saline (PBS; pH 7.4) was used as hydration solution. The obtained micelle solution was size-controlled by miniextruder and then purified through sepharose (CL-4B) to remove the remaining free CUR. For the preparation of unmodified polymeric micelles containing CUR (PM-CUR), an identical procedure was conducted, replacing apamin-PEG-DSPE with an equivalent amount of mPEG-DSPE. The preparation of polymeric micelles with fluorescent reagents was performed by following a procedure identical to 3211

dx.doi.org/10.1021/mp500393m | Mol. Pharmaceutics 2014, 11, 3210−3222


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were excised. The near-infrared fluorescence signal intensities were measured in each tissue. Curcumin and fluorescent probe RhB were coencapsulated in micelles and used in the confocal microscopy study. KM mice were divided into 2 groups, with one group undergoing administration of 0.1 mL of apamin-PM-CUR/RhB (CUR, 10 μg; RhB, 13 μg) via tail vein and the other receiving PM-CUR/RhB. Animals were sacrificed 4 h following administration and immediately perfused with 4% paraformaldehyde (PFA). The spinal cords were explanted, kept in 4% PFA at 4 °C for 24 h, and subsequently cryoprotected in 30% sucrose in PBS. Cross sections of the spinal cord were cryosectioned (each section 10 μm in thickness) for DAPI staining. The fluorescent images of the spinal cord sections (T7−T10) were analyzed using a confocal microscope (LSM700, Carl Zeiss, Germany). The biodistribution study was carried out in KM mice randomly divided into two treatment groups, receiving apaminPM-CUR or PM-CUR. In each group, mice were sacrificed by isoflurane inhalation 0.5, 1, 2, 4, 8, and 12 h following administration and tissues were collected (n = 6 at each time point). Tissue samples were weighed and homogenized in PBS. CUR was extracted from the collected tissue samples and quantified by HPLC. 2.5. Establishment and Evaluation of SCI Animal Models. Animal models were established according to Allen’s weight dropping (WD) at thoracic spinal cord level.37 A total of 180 KM mice, weighing 18−22 g (20 g on average), were used to construct a mouse model of traumatic spinal cord injury (SCI). The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (5 mL/kg). A series of operations were performed: a dorsal midline incision was made, and the dorsal face of T8 was exposed. The spinous process and lamina were removed, and a circular region of dura, approximately 2.5 mm in diameter, was exposed. After stabilization of the vertebral column, a 3 g weight was dropped 5.0 cm onto the exposed dura. After injury, the overlying skin was closed.38 Each of the treated mice were housed individually in cages and administered penicillin 20,000 U by intramuscular injection twice daily. Moreover, animals received urination assistance 2− 4 times per day by squeezing the bladder until the micturition reflex recovered. 2.6. In Vivo Pharmacodynamics Study. SCI mice (n = 40) were randomly divided into four groups: group 1 were injected with physiological saline daily for 1 week; group 2 received 30 mg/kg of MP as a bolus injection within the first 8 h after SCI and a constant-rate infusion over the following at least 23 h,39 while groups 3 and 4 were administered apaminPM-CUR or PM-CUR (5 mg/kg of curcumin) via tail vein daily for 1 week. The recovery of the SCI mice was observed 0, 6, 12, and 24 h following SCI, and 3, 7, 14, 28, 56, and 168 days thereafter) using the Basso mouse scale (BMS) scoring system,40,41 measurement of motor evoked potentials (MEP),42 and observation of the survival of treated animals. Hematoxylin and eosin (H&E) and horseradish peroxidase (HRP) retrograde staining were also used to evaluate the recovery of SCI mice. H&E staining was used to evaluate the morphological structure of the spinal cord tissue, while HRP retrograde staining was used to track the peripheral nerve fibers of the CNS. The staining approaches used were previously shown to be capable of evaluating the structural integrity and functional completeness of spinal cord axonal.43 Following the observation period (24 weeks following administration), animals in each of the four groups were

that used for CUR-loaded polymeric micelles, replacing CUR with DiR. 2.3.2. Characterization of Polymeric Micelles. The particle size of apamin-PM-CUR was measured by photon correlation spectroscopy using a Zetasizer Nano ZS (Malvern, Worcestershire, U.K.) at 25 °C. To confirm the identity of the bioactive ligand displayed on the external shell of obtained micelles, X-ray photoelectron spectroscopy (XPS) analysis (imaging photoelectron spectrometer, Axis Ultra, Kratos Analytical Ltd.) was performed. The chemical surface composition of the freeze-dried nanocarriers was investigated to check the presence of nitrogen in the ligands introduced via the grafted NHS-PEG-DSPE.32 X-ray diffraction (X-RD) spectrometry was used to characterize the drug encapsulation of apamin-PM-CUR, since the crystal diffraction peak of curcumin will disappear after packing into micelles.33 CUR, apamin-PM, its graft copolymer apaminPM-CUR, and the physical mixture of CUR and apamin-PM at the same material ratio were analyzed in parallel by X-RD spectrometry using an XD-3A powder diffraction meter with Cu Kα radiation in the range 10−50° (2θ) at 40 kV and 30 mA. In vitro release of CUR from apamin-PM-CUR and PM-CUR was evaluated by dynamic dialysis. Briefly, a sample of CUR formulation was mixed with 1 mL of buffer solution, placed into a dialysis bag (molecular weight cutoff 10000 Da), and incubated with shaking in 150 mL of release medium (PBS at pH 7.4 with 2% sodium dodecyl sulfate (SDS) at 37 °C). Samples (0.3 mL) were taken at predetermined time intervals (0.5, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 48, 72, and 96 h) from the release medium over a period of 24 h and replaced by the equivalent volume of fresh medium. The concentration of CUR was determined by HPLC. 2.4. Evaluation of Targeting Ability. 2.4.1. In Vivo Imaging of Apamin-Mediated Targeted Delivery of Macromolecules. The apamin-conjugated macromolecules were prepared by incubation of FITC-labeled streptavidin with biotinylated apamin and subsequent purification through dialysis.34,35 The tissue distribution of apamin-conjugated macromolecules (FITC-SA-biotinylated apamin) and control macromolecules (FITC-labeled streptavidin, FITC-SA) was detected by fluorescence imaging. Mice were sacrificed 0.5, 1, 2, and 4 h following injection of a 0.1 mL formulation of FITC-SA-biotinylated apamin or FITC-SA, and the brain, spinal cord, heart, liver, spleen, and kidneys were excised and scanned using an in vivo imaging system (FX-Pro, BRUKER, Germany), with an excitation bandpass filter at 470 nm and an emission wavelength of 530 nm. 2.4.2. In Vivo Imaging of Apamin-Mediated Targeted Delivery of Micelles. Thirty-five KM mice were randomly divided into 5 groups, and 3 of the groups were injected with free DiR, PM-DiR, and apamin-PM-DiR via the tail vein, respectively. The remaining two groups were pretreated by injection of 0.1 mL of free apamin (40 μg) or an equimolar amount of dequalinum chloride (10 μg), as a selective blocker of apamin-sensitive K+ channels,36 1 h prior to the administration of apamin-PM-DiR. Mice were anesthetized and scanned at 0.5, 1, 2, 4, 8, 12, and 24 h postinjection using an in vivo imaging system (FX-Pro, BRUKER) with an excitation bandpass filter at 730 nm and the emission wavelength of 790 nm. Hair was removed from the mice using 8% sodium sulfide solution. Following imaging, mice were sacrificed at the same time points, and the brain, spinal cord, heart, liver, spleen, lungs, and kidneys 3212



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Figure 1. Characterization of apamin-PEG-DSPE and polymeric micelles. The MALDI-TOF MS analysis of NHS-PEG-DSPE and apamin-PEGDSPE. The arrow indicates the peak corresponding to the targeting compound. The molecular weight of apamin-PEG-DSPE was determined to be approximately 5542 Da (A). The 1H NMR spectra of NHS-PEG-DSPE and apamin-PEG-DSPE (B). The results of general XPS survey scans of apamin-PM (C). The nitrogen signals of apamin-PM and PM (D). X-ray diffraction profiles of curcumin (CUR)-loaded apamin-PM-CUR, unloaded apamin-PM, CUR alone, and the physical mixture of unloaded apamin-PM and CUR at approximately the same ratio as in apamin-PM-CUR (E). In vitro CUR release from apamin-PM-CUR and PM-CUR in PBS pH 7.4 with 2% SDS at 37 °C. Data are presented as means ± SD (n = 6) (F). a.u., arbitrary units.

divided into two subgroups, with half of the animals sacrificed and immediately perfused with 4% PFA. The T7−T10 segments of spinal cords extracted from these animals were explanted, kept in 4% PFA at 4 °C for 24 h, and later cryoprotected in 30% sucrose in PBS.44 Cross sections of the spinal cord through the lesion site were cryosectioned (10 μm in thickness) for

H&E staining. The other half of the treated animals were used for HRP staining. Mice in these subgroups were microinjected HRP in the inferior extremity of SCI sites and sacrificed 48 h later, allowing enough time for nerve cells to take up the HRP. SCI sites and their superior extremities were dissected and the collected tissues kept in 4% PFA at 4 °C for 24 h and 3213



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subsequently cryoprotected in 30% sucrose in PBS. Longitudinal sections of the spinal cord T7−T10 were cryosectioned (10 μm in thickness) for 3,3′,5,5′-tetramethylbenzidine (TMB) coloration according to the method previously described by Weinberg et al.43 2.7. Potential Toxicity Tests. The primary test of LD50 (lethal dose for 50% of the animals) for apamin and apaminconjugated PEG-DSPE was calculated by the Karber method.45 192 KM mice were randomly divided into 16 groups: 8 apamintreated groups (2, 3, 4, 5, 7, 10, 15, and 20 mg/kg) and 8 polymer-treated groups (55, 76, 106, 146, 203, 282, 391, and 542 mg/kg). All animals received treatments via tail vein injection, and toxicity was evaluated 2, 4, 6, 8, and 12 h following administration and 1, 3, 7, and 14 days postinjection. The mortality and symptoms of survivors were recorded in each treatment group. For EEG monitoring, 78 mice were divided into 13 groups: 6 apamin-treated groups (2, 4, 10, 20, 100, and 200 mg/kg), 6 polymer-treated groups (containing apamin equivalent to 2, 4, 10, 20, 100, and 200 mg/kg), and a saline-treated control group, with n = 6 in each group. The electrodes were surgically implanted in a sterile environment using previously described methods.46 Briefly, the mouse head was fixed in the stereotaxic frame to keep the horizontal orientation. The electrodes were positioned at the following coordinates: (+)-lead, caudal of bregma 2 mm, lateral of bregma 2 mm (right hemisphere), dorsoventral (depth) 2 mm (relative to the dorsal surface of the calvaria and corresponding to the hippocampus as the final targeting region); (−)-lead, caudal of bregma 6.2 mm, lateral of bregma 0 mm, dorsoventral 0 mm (epidural reference electrode localized on the cerebellum). The electrodes were fixed following positioning, and the scalp was closed. All animals were allowed a minimum of 3 weeks for recovery from surgery before any experimental procedures or recordings were performed. 2.8. Statistical Analysis. Survival data were analyzed using a log-rank test and presented using Kaplan−Meier plots. Student’s t test was employed for comparing two groups, and one-way analysis of variance (ANOVA) test was applied for comparisons between multiple groups. P < 0.05 was considered statistically significant.

Table 1. Characterization of Polymeric Micelles (n = 3) polymeric micelles Z-average (nm) apamin-PM400-DiR apamin-PM200-DiR apamin-PM100-DiR apamin-PM50-DiR PM50-DiR apamin-PM50-CUR apamin-PM50 a

404.21 201.33 103.67 43.84 51.12 53.33 50.03

± ± ± ± ± ± ±

1.94 3.51 2.49 1.50 2.02 1.06 1.07

PDIa 0.201 0.158 0.165 0.100 0.109 0.200 0.177

± ± ± ± ± ± ±

0.22 0.03 0.03 0.01 0.04 0.01 0.03

encapsulation efficiency (%) 78.06 79.11 78.53 75.63 76.34 79.00

± ± ± ± ± ±

0.12 0.90 0.87 1.23 0.73 1.17

PDI, polydispersity index.

Figure 2. Ex vivo fluorescent images of tissues dissected from mice administered FITC-SA (a) or FITC-SA-biotinylated apamin (b).

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Apamin and Apamin-Modified Material. Bee venom has a long history of medicinal use around the world, with recorded use since ancient times. Currently, bee venom is approved by the FDA for use in allergy desensitization, with a number of other clinical uses explored. For example, an injection of apitoxin has been used clinically for treatment of rheumatic arthritis, Parkinson’s disease, and peripheral neuritis in China and other countries.47,48 The active components of bee venom are primarily proteins and peptides, among which apamin comprises about 2−3% by weight of the total dried venom mass. Apamin, an 18-amino acid peptide, is the smallest known natural polypeptide which can penetrate the blood brain barrier. Moreover, apamin receptors were found to be highly expressed in both dorsal and lumbar spinal cord.49 Therefore, a possibility exists that apamin can be clinically used for targeted therapy of CNS diseases. According to the crystal structure of apamin presented in the graphical abstract,50 the side-chain amino group of the fourth lysine was solvent-exposed and located distal to the main structure, which may qualify it as the conjugation site for further modification. The targeting compound comprising

apamin-PEG-DSPE was synthesized by the linkage of apamin to the NHS-PEG-DSPE through a nucleophilic substitution reaction which forms the amide bond. As shown in Figure 1A, the experimental molecular weights of NHS-PEG-DSPE and apamin-PEG-DSPE were determined by MALDI-TOF MS to be 3523 and 5542 Da, respectively. These values are in accordance with the theoretical molecular weight, confirming the identity of the synthesis product. The 1H NMR spectra of NHS-PEG-DSPE and apamin-PEG-DSPE in CDCl3 are shown in Figure 1B. The disappearance of δ2.60 in NHS-PEG-DSPE indicates that the conjugation was successful.51 3.2. Characterization of Polymeric Micelles. The characteristics of blank micelles and CUR- and DiR-loaded polymeric micelles modified with apamin are summarized in Table 1. All prepared polymeric micelles had uniform particle size with narrow distribution. The apamin-PM400-DiR, apaminPM200-DiR, apamin-PM100-DiR, and apamin-PM50-DiR refer to fluorophore encapsulated micelles with different particle sizes (400, 200, 100, 50 nm, respectively), which were evaluated to investigate the influence of particle size on in vivo distribution of micelles. 3214



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Figure 3. Effect of particle size and the amount of peptide ligand on the targeting efficiency of apamin-PM. Ex vivo fluorescent imaging study (A) and the semiquantitative results of ex vivo fluorescence imaging (B): 400 nm, 5 mol % (a), 200 nm, 5 mol % (b), 100 nm, 5 mol % (c), 50 nm, 5 mol % (d), 50 nm, 0.5 mol % (e), and 50 nm, 0.05% (f).

From our investigation, approximately 80% of CUR and 75% of DiR can be successfully loaded into polymeric micelles (Table 1). Additionally, modifications of the polymeric micelles with the ligand showed no obvious effect on the drug encapsulation efficiency. No drug leakage and no significant changes in the particle size, polydispersity index, and charge were detected in APM-CUR following 2 months of storage at 4 °C. X- PS was used for the detection of nitrogen in apamin-PEGDSPE. The decomposition of the N 1s peak (Figure 1C) confirmed the presence of amine bonds at 399 eV, amide bonds around 400 eV, and protonated nitrogen around 401 eV.32 No peaks at these three energy levels were observed in PEG-DSPE (Figure S1 in the Supporting Information). As evidenced by the absence of nitrogen in the native copolymers and blank samples (Figure 1D), the nitrogen detected in the apamin-PEG-DSPE samples likely reflects the presence of covalently bound peptides. X-ray diffraction profiles of drug-loaded micelles (apaminPM-CUR) and the physical mixture of CUR and blank micelle (apamin-PM) at the same drug-to-material ratio are shown in Figure 1E. CUR has two high intensity reflections that fall at 2θ = 32°, 2θ = 46°, and apamin-PM has two reflections which fall at 2θ = 19°, 2θ = 33°, while all four of these reflections were detected in the physical mixture. Analysis of apamin-PM-CUR detected reduced intensity of all peaks, suggesting that the encapsulation of curcumin in micelles was successful.

In vitro drug release profiles of curcumin from apamin-PMCUR and PM-CUR in PBS at pH 7.4 with 2% SDS at 37 °C are shown in Figure 1F. No significant differences were observed between apamin-PM-CUR and PM-CUR at various time points. This result suggests that the release of CUR polymeric micelles is not altered by modification with apamin. 3.3. Apamin-Mediated Targeting of Biomacromolecules and Nanoparticles to CNS. The apamin-mediated drug targeting to CNS was initially confirmed using steptavidin as a model drug biomacromolecule. The apamin conjugate was assembled using the biotin−streptavidin system. As shown in Figure 2, the fluorescence intensity in the spinal cord and brain of the biomacromolecule (FITC-labeled streptavidin) control group is marginal while the fluorescence in animals treated with apamin bioconjugate is remarkably increased at all detected time points, suggesting targeted delivery of biomacromolecule mediated by apamin. A small enough particle size with adequate modified ligands is the prerequisite for the construction of nanosized active targeting drug delivery systems. Therefore, the influence of particle size and the amount of apamin-PEG-DSPE on CNS targeting of APM was sequentially investigated. Using 5 mol % apamin-PEG-DSPE material composition, 50 nm sized micelles showed the highest target tissue distribution of fluorescence probes both in vivo and ex vivo, as compared to the 100, 200, 3215



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mice. In electrophysiological experiments, the incubation period of MEP-N1 peaks of normal mice at each observation point remained at around 2 ms, while the condition was drastically different for the SCI mice. The initial incubation period of MEP-N1 peak of SCI mice was more than 10 ms. Three days following injury, the period prolonged to 13 ms. Twenty-four weeks later, no identifiable MEP-N1 peak was found in the few surviving animals. The results of H&E and HRP staining also differentiate the SCI model mice and normal mice, with central cavitation and cells of incomplete structures observed in the injured spinal cord section, with lack of myelin sheath in the visual fields. It was confirmed that SCI mice cannot recover without therapy within the observation time. Since SCI has been reported to disrupt the blood−spinal cord barrier over a period of time, a comparison of tissue distribution was performed, evaluating the distribution of peptide-modified micelles and unmodified micelles in SCI model mice using in vivo fluorescence imaging assay.53 The model mice were established and used within 1 h in case the barrier permeability remained elevated at maximal levels. As shown in Figure 6, after intravenous administration of same doses of fluorophore-loaded micelles, APM-DiR showed significant targeted accumulation in SCI model mice, as compared to animals treated with PM-DiR. Only a slight difference in fluorophore signal in the target tissue was observed between the PM-treated model mouse group and the PM-treated normal mouse group, demonstrating the potential importance of active-targeted drug delivery for SCI therapy. After 1 week of continuous treatment with curcumin formulations, praxiological evaluation of animal models has shown that animals treated with PM-CUR experienced marginal improvement over those treated with physiological saline. Animals treated with apamin-PM-CUR and MP exhibited drastic improvements in functional outcomes. Specifically, animals treated with apamin-PM-CUR scored significantly higher on the BMS, as assessed 1, 2, 4, 8, and 24 weeks following treatment, as compared to all other treatment groups. In contrast, animals treated with PM-CUR showed no significant differences compared to animals treated with physiological saline. The outcomes measured with other scales showed that animals treated with apamin-PM-CUR scored significantly higher than PM-CUR and somewhat higher than MP-treated animals, with representative changes in BMS subscore in SCI animals presented in Figure 7A. After 24 weeks, almost all mice treated with apamin-PM-CUR exhibited good recovery. The scale reached almost 10, which may suggest that apamin-PM-CUR is helpful for the recovery following SCI in mice. Measurement of MEP was used for intraoperative monitoring of the functional integrity of the pyramidal tract.42 Figure 7B shows the incubation period of the MEP-N1 peak of each group at a range of time points (0.6 and 12 h, 1 and 3 days, 1, 2, 4, 8, and 24 weeks). All 4 groups had no detectable MEP-N1 at 0 and 6 h following administration. At 12 h, the incubation periods of N1 peak were almost the same (10 ms) in all 4 treatment groups, indicating that the animal model of SCI was successfully induced with uniform severity. At the third day, the incubation period of the MEP-N1 peak of apamin-PM-CUR and MP was substantially diminished and showed a significant difference compared to the period measured in animals treated with physiological saline at 8 weeks post-treatment. Displaying a similar trend as observed in BMS scores, both apaminPM-CUR and MP remarkably improved the recovery of spinal cord injury, which was much better than the recovery following PM-CUR. Apamin-PM-CUR was found to be more effective in inducing spinal cord recovery than methylprednisolone.

and 400 nm particles. At a mean particle size of 50 nm, micelles with 5 mol % peptide ligand demonstrated the best targeting capacity, while micelles with 0.5 mol % of modified apamin exhibited a strong fluorescence signal in spinal cord and brain tissues, approximately equal to that observed with the former one (Figure 3). When considering potential applications, apamin-PM particles of 50 nm size with 0.5 mol % targeting ligand were chosen for further study. Curcumin and rhodamine were coencapsulated by APM and injected into KM mice. The colocalization of two fluorescent probes in the histological spinal cord sections indicated efficient targeted delivery of polymeric micelles (Figure 4). Furthermore, when given as a

Figure 4. Confocal microscopy images of spinal cords of mice administered apamin-PM-CUR/RhB (A) or PM-CUR/RhB (B) (all images shown at ×200 magnification).

single dose of 5 mg/kg of encapsulated curcumin, APM showed significantly improved drug distribution in both spinal cords and brains of mice, compared to the unmodified micelles (Figure S2 in the Supporting Information). Ion channels are promising therapeutic targets for neurological diseases and may also serve as receptors or cell recognition molecules on target cell surfaces for active targeted drug delivery.52 As shown in Figure 5, preinjection of either free apamin or an equal amount of dequalinium chloride 1 h prior to APM can significantly reduce the targeted distribution of encapsulated fluorophore, suggesting the existence of a specific interaction between apamin and SK channels in the recognition process. 3.4. In Vivo Pharmacodynamics Study. The SCI mouse model was implemented using a standard process described above, with the success confirmed by a series of characterizations at multiple levels (Figure S3 in the Supporting Information). Notable differences were found in animal survival and BMS scores between the SCI model animals and normal 3216



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Figure 5. In vivo fluorescent images of five groups of mice treated by DiR-loaded formulations administered via tail vein at 0.5, 1, 2, 4, 8, 12, and 24 h postinjection (A). Ex vivo fluorescent images of brains, spinal cords, and other major organs dissected from animals of the five treatment groups at the same time points postinjection (B). Animals were administered DiR (a), PM-DiR (b), apamin-PM-DiR with pretreatment with apamin 1 h prior to the injection (c), apamin-PM-DiR with pretreatment with dequalinum chloride 1 h prior to the injection (d), and apamin-PM-DiR (e).

and dissected spinal cord samples immediately cryosectioned (10 μm in thickness) in cross section for H&E staining (Figure 8A) and in longitudinal section for HRP staining (Figure 8B). The changes induced by spinal cord injury were observed under optical microscope at ×400 magnification). As shown in Figure 8A, animals treated with PM-CUR lose the typical tissue structure. Most cell nuclei were dissolved, cytoplasm appeared cloudy, and a number of large cavities formed in the cytosol. In comparison, no noticeable improvement was observed in the animals treated with physiological saline. In contrast, in the tissues obtained from the animals administered MP and apamin-PM-CUR, the spinal cord tissue structure was clearer than in other groups. Neurons were normal in appearance, with maintained membrane integrity, visible cytoplasmic Nissl bodies, and glial scar tissue with regular appearance. Taken together, these results suggest that

We reviewed animal survival data in an effort to evaluate any potential improvements in overall animal health (Figure 7C). In animals treated with MP or apamin-PM-CUR, survival rates were 90% and 100%, respectively, 24 weeks following SCI induction. In comparison, animals treated with PM-CUR and physiological saline showed dramatic mortality, with survival rate below 30%, during the same period. Additionally, no obvious recovery of model mice was detected in an independent assay performed within 4 weeks when animals were administered polymers alone (apamin-modified PEGDSPE or PEG-DSPE) at the doses equal to those used in apamin-PM-CUR treatment (Figure S4 in the Supporting Information). These results demonstrate the significant efficacy of targeted delivery of curcumin against spinal cord injury. 3.5. Histopathology and Immunohistochemistry. Twenty-four weeks after administration, animals were sacrificed 3217



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Figure 6. Ex vivo fluorescent images of brains, spinal cords, and other major organs dissected from SCI animals and normal mice administered apamin-PM-CUR and PM-CUR via tail vein injection at 0.5, 1, 2, 4, 8, 12, and 24 h postinjection. Normal mice were administered apamin-PM-DiR (a) and PM-DiR (b). SCI mice administered PM-DiR (c) and apamin-PM-DiR (d).

apamin-PM-CUR helped relieve secondary spinal cord injury and promoted neuronal regeneration. Figure 8B shows the results of HRP retrograde staining. In animals administered PM-CUR, the myelin sheath was faintly colored, cellular arrangement was disorderly, anatomical contact was interrupted, and the formation of large cavities was not notably different from the tissues obtained from saline-treated animals. However, in animals treated with MP or apaminPM-CUR, cells were stained clearly and arrayed in a chainlike arrangement. Very few cavities were observed in the apaminPM-CUR treatment group. In these two groups, cells exhibited better regeneration of neural connections and better recovery after SCI compared to the former two groups. The results provide strong evidence suggesting that SCI mice recovered on the histological level following treatment with MP and apamin-PM-CUR. 3.6. Toxicity Tests. For the primary toxicity test, the LD50 values for apamin and apamin-conjugated PEG-DSPE were determined to be 10.3 and 283.0 mg/kg (containing apamin equivalent to 103 mg/kg), respectively. Tables 2 and 3 present the lethality in SCI mice over time and the symptoms found in survivors following treatment.

Figure 7. Evaluation of functional recovery of SCI mice based on the Basso mouse scale (BMS) scoring (A), motor evoked potentials (MEP) detection (B), and animal survival observation (C). The recovery of the SCI mice was observed at various time points. The results were performed across four groups: (a) methylprednisolone (black, −■−), (b) apamin-PM-CUR (red, −●−), (c) PM-CUR (blue, −▲−), (d) physiological saline (green, −▼−). Data represented as mean ± SD (n = 10). *P < 0.1, **P < 0.01, ***P < 0.001 vs saline; # P > 0.1, ##P < 0.01 vs apamin-PM-CUR. 3218



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Figure 8. Images of H&E staining (A) and HRP staining (B) of spinal cords of mice at 24 weeks after SCI for methylprednisolone (a), apamin-PMCUR (b), PM-CUR (c), physiological saline (d), and normal animal (e) (×400).

Figure 9. Deep intracerebral (hippocampal) EEG recordings of apamin (2, 4, 10, 20, 100, and 200 mg/kg) and apamin-PEG-DSPE (containing apamin equivalent to 2, 4, 10, 20, 100, and 200 mg/kg).

Site-modification of apamin has been reported to decrease the neurotoxic effect.54,55 In the current evaluation, the conjugation of PEG-DSPE showed significant detoxification of apamin by lowering the LD50 value 10-fold while maintaining the targeting to the CNS. There has been a long history of human therapeutic use of bee venom, in a “fighting fire with fire” conceptual approach. Apamin was proven to have therapeutic potential when administered at proper doses, with applications including treatment of ataxia, epilepsy, memory disorders, and possibly schizophrenia and Parkinson’s disease,

EEG monitoring of brain function in treated mice presented similar trends. In the free peptide group, decreased amplitude and increased frequency of the EEG signals were found following a dose of 10 mg/kg. More drastic signal changes occurred at doses greater than 100 mg/kg, which led to no survivors. In animals treated with apamin-conjugated polymer, no deaths were observed at apamin doses below 20 mg/kg. EEG signals recorded in mice administered polymers at all doses remained in a normal range, except in animals administered the highest two doses (Figure 9). 3219



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Notes

Table 2. Death Times and Symptoms of Mice after Administration Apamin (n = 12 per Treatment Dose)a iv dose (mg/kg)

death time (min)

symptoms in survivors

2 3 4 5 7 10 15 20

no death 25 10, 20 15, 15, 20 10, 15, 20, 30 10, 10, 15, 20, 30 5, 5, 10, 15, 15, 20, 20, 30 5, 5, 10, 10, 10, 15, 15, 15, 20, 25, 30

O100% O92% O67%, S17% O50%, S17%, SS8.3% O16%, S42%, SS8.3% S42%, SS17% SS17%, SSS17% SSS8.3%

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors are grateful for financial support from the National Natural Science Foundation of China (No. 81102404 and No.21272187), Program for Innovative Research Team in University of Chongqing (2013), and the Fundamental Research Funds for the Central Universities (XDJK2013A015, 2362014xk07).

■

ABBREVIATIONS USED APM, apamin-modified polymeric micelles; BMS, Basso mouse scales; CNS, central nervous system; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole; DiR, 1,1′-dioctadecyl-3,3,3,3′tetramethylindotricarbocyanine iodide; DMF, N,N-dimethylformamide; EEG, electroencephalogram; EPR, enhanced permeability and retention; FITC, fluorescein isothiocyanate; H&E, hematoxylin and eosin; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; KM mouse, Kunming mouse; MALDI-TOF, matrix assisted laser desorption/ionization time-of-flight; MEP, motor evoked potentials; MP, methylprednisolone; MS, mass spectrometry; NHS-PEGDSPE, 3-(N-succinimidyloxyglutaryl)aminopropyl, polyethyleneglycol-carbamyl distearoylphosphatidyl-ethanolamine; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; PFA, paraformaldehyde; RhB, rhodamine B; SCI, spinal cord injury; SDS, sodium dodecyl sulfate; TMB, 3,3′,5,5′-tetramethylbenzidine; X-RD, X-ray diffraction spectrometry; XPS, X-ray photoelectron spectroscopy

a

LD50: 10.3 mg/kg. Numbers indicate death times (min); S, SS, SSS indicate increasing severity of symptoms; O indicates no symptoms.

Table 3. Death Times and Symptoms of Mice after Administration of Apamin-PEG-DSPE (n = 12 per Treatment Dose)a iv dose (mg/kg)

death time (min)

symptoms in survivors

55 76 106 146 203 282 391 542

no death 15 20, 30 15, 20, 25 15, 15, 30, 4320 10, 15, 20, 30, 1440 5, 10, 10, 15, 20, 25, 35 5, 5, 10, 10, 10, 15, 15, 20, 20, 25

O100% O92% O67%, S17% O50%, S17%, SS8.3% O16%, S42%, SS8.3% S42%, SS17% S17%, SSS25% SS8.3%, SSS8.3%

■

a

LD50: 283.0 mg/kg. Numbers indicate death times (min); S, SS, SSS indicate increasing severity of symptoms; O indicates no symptoms.

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among others.56,57 In the present study, a single dose of peptide copolymers was equivalent to 0.15 mg/kg of apamin, which is about 1/700 of the determined LD50 value, suggesting promising potential for its clinical application.

4. CONCLUSION In summary, we constructed an efficient CNS-targeting drug delivery system specific for the spinal cord using apamin as a targeting ligand. The optimized 50 nm sized micelles with 0.5 mol % targeting ligand significantly enhanced the accumulation of model drug CUR in spinal cord and showed high efficacy in tissue repair following SCI. No toxicity was observed at the treatment dose, demonstrating the very promising potential of apamin-mediated targeted therapy for SCI and other CNS diseases.

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ASSOCIATED CONTENT

S Supporting Information *

Results of general XPS survey scans of PM (Figure S1). Tissue distribution of CUR in mice administered apamin-PM-CUR and PM-CUR (Figure S2). Evaluation of spinal cord injury (SCI) mouse model using Basso mouse scale scoring, motor evoked potential detection, and observation of survival in treated animals (Figure S3). Evaluation of functional recovery of spinal cord injury model mice based on the Basso mouse scale scoring and observations of animal survival (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-23-68251225. Fax: +86-23-68251225. E-mail: [email protected]. 3220



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