Angewandte Nanomedicine Do:10.1002/anie201100875 Micelle-Based Brain-Targeted Drug Delivery Enabled by a Nicotine Acetylcholine Receptor ligand x Changyou Zhan, Bian Li, Luojuan Hu, Xiaoli Wei, Linyin Feng, Wei Fu, and Weiyue Lu* The blood-brain barrier(BBB)is the key challenge in the analysis, a and confirmed by using immunocytochemical development of drugs for diseases of the central nervous staining(Figure S8 in the Supporting Information, the a7 system(CNS). The BBB prevents drugs or drug delivery neuronal nAChR is richly expressed in primary brain systems from reaching the site of disease because of tight capillary endothelial cells, and is thus ideally suited for junctions and lack of fenestration. I To circumvent this candoxin-mediated, brain-targeted drug delivery. For this roblem, the receptors that are highly expressed on the study, we designed and evaluated three short peptides derived capillary endothelium of the brain, such as nicotine acetylcho- from the loop II region of candoxin, FKESWREARG line receptors(nAChRs), have been exploited to facilitate TRIERG (CDX), SWREARGTRI (Pocket_CDX), and BBB crossing and intracranial transport of drug delivery disulfide bridged CFKESWREARGTRIERGC systems. 2) nAChRs are ligand-gated ion channels that are (Cyclo_CDX) expressed mainly at the neuromuscular junction of the CNS, hether or not the candoxin-derived including the brain capillary endothelial cells. 3I The extensive peptides are capable of interacting with rat neuronal expression of nAChRs in the brain and susceptibility to the nAChRs, we performed a competitive binding assay where inhibition by peptide neurotoxins and neurotropic viral different concentrations of peptide competed for receptor proteins endow them with the ability to mediate peptide- binding with radiolabeled I-a-bungarotoxin, which is a based transvascular delivery of various therapeutic agents to potent antagonist of a7 neuronal nAChRs.S)All three the brain 24.4 Herein, we report the design of a 16-residue peptides functioned as competitive antagonists of neuronal peptide, derived from the loop II region of the snake neuro- nAChRs in a dose-dependent manner(Figure S2). CDX toxin candoxin, that binds to nAChRs with high affinity. This displayed a K, value of 0. 187 uM, which is approximately 20- peptide, termed CDX, enabled drug delivery to the brain 40 times lower than those of Pocket_CDX and Cyclo_CDX when conjugated to paclitaxel-loaded micelles. As a result, (Table 1). Not surprisingly, CDX is substantially less poter tumor growth in intracranial glioblastoma bearing mice was than candoxin in nAChRs binding. The difference in potency inhibited and their survival was prolonged is likely attributable, at least in part, to a loss of entropy for Snake neurotoxins are members of the"three-finger CDX, as it is unstructured in aqueous solution, as indicated by oxin"superfamily characterized by three adjacent loops circular dichroism spectroscopic analysis(Figure $3) arranged in a flat, leaflike structure. 5I These toxins are known to bind through the second loop to nAChRs with high affinity Table 1: Experimentally determined versus predicted free energies of and selectivity. Candoxin from the Malayan krait Bungarus binding of all synthetic peptides with a7 neuronal nAChR andidus consists of a single polypeptide chain of 66 amino neuronal nAChRs in nanomolar concentrations with poor Peptideb36log KQ). acid residues with five disulfide bridges, and antagonizes a7 uM] Experimental△ G Estimated△G reversibility. I As was shown previously by western blot [kcal mol- 0.187 Pocket_CDX -8.17 B X. Wei. Dr. W. Fu. Prof. yolo_CDX 7.11 7.85 School of Pharmacy Key Laboratory of Smart Drug Delivery Ministry of Education PLA, Fudan University Shanghai 201203(P R China) Fax:(+86)2-51980090 Based on the structure of candoxin(PDB code: 1JGK) E-mail:weifuuh@gmail.com elucidated by NMr spectroscopy, and the crystal structure of wylu@shmu.edu.cn acetylcholine-binding protein(AChBP; PDB code: 1YI5 Dr L Hu. Prof Lf that shares a high degree of State Key Laboratory of Drug Research nce identity with the hanghai Institute of Materia Medica extracellular ligand-binding domain of a7 neuronal nAchRs. Shanghai 201203(PR China) we conducted molecular modeling and docking studies to [] These authors contributed equally to this work. better understand the interactions of the a7 nachr with the [*] This work was supported by National Basic Research Program of CDX peptides. All three peptides adopt hairpinlike confor hina(973 Program)2007CB935800 and 2010CB934000, the"Key mations that feature a unique electrostatic potential witi New Drug Creation Program"2009ZX09310-006, and Shanghai segregating cationic and anionic patches(Figure S5, S6). The Nanotechnology Program(0953nm03300 highly charged tip of CDx penetrates deeply into the O) Supporting information for this article is available on the www hydrophobic binding pocket formed by two adjacent subunits underhttp://dx.doi.org/10.1002/anie.201100875 of the a7 nAChR, and the binding is dominated by cation-t Chem. Int. Ed. 2o11, 5o, 1-5 o 2011 Wiley-VCH Verlag GmbH Co KGaA, Weinheim These are not the final page numbers WLEY咱
Nanomedicine DOI: 10.1002/anie.201100875 Micelle-Based Brain-Targeted Drug Delivery Enabled by a Nicotine Acetylcholine Receptor Ligand** Changyou Zhan, Bian Li, Luojuan Hu, Xiaoli Wei, Linyin Feng, Wei Fu,* and Weiyue Lu* The blood–brain barrier (BBB) is the key challenge in the development of drugs for diseases of the central nervous system (CNS). The BBB prevents drugs or drug delivery systems from reaching the site of disease because of tight junctions and lack of fenestration.[1] To circumvent this problem, the receptors that are highly expressed on the capillary endothelium of the brain, such as nicotine acetylcholine receptors (nAChRs), have been exploited to facilitate BBB crossing and intracranial transport of drug delivery systems.[2] nAChRs are ligand-gated ion channels that are expressed mainly at the neuromuscular junction of the CNS, including the brain capillary endothelial cells.[3] The extensive expression of nAChRs in the brain and susceptibility to the inhibition by peptide neurotoxins and neurotropic viral proteins endow them with the ability to mediate peptidebased transvascular delivery of various therapeutic agents to the brain.[2d, 4] Herein, we report the design of a 16-residue peptide, derived from the loop II region of the snake neurotoxin candoxin, that binds to nAChRs with high affinity. This peptide, termed CDX, enabled drug delivery to the brain when conjugated to paclitaxel-loaded micelles. As a result, tumor growth in intracranial glioblastoma bearing mice was inhibited and their survival was prolonged. Snake neurotoxins are members of the “three-finger toxin” superfamily characterized by three adjacent loops arranged in a flat, leaflike structure.[5] These toxins are known to bind through the second loop to nAChRs with high affinity and selectivity.[6] Candoxin from the Malayan krait Bungarus candidus consists of a single polypeptide chain of 66 amino acid residues with five disulfide bridges, and antagonizes a7 neuronal nAChRs in nanomolar concentrations with poor irreversibility.[7] As was shown previously by western blot analysis,[3d] and confirmed by using immunocytochemical staining (Figure S8 in the Supporting Information), the a7 neuronal nAChR is richly expressed in primary brain capillary endothelial cells, and is thus ideally suited for candoxin-mediated, brain-targeted drug delivery. For this study, we designed and evaluated three short peptides derived from the loop II region of candoxin, FKESWREARGTRIERG (CDX), SWREARGTRI (Pocket_CDX), and disulfide bridged CFKESWREARGTRIERGC (Cyclo_CDX). To investigate whether or not the candoxin-derived peptides are capable of interacting with rat neuronal nAChRs, we performed a competitive binding assay where different concentrations of peptide competed for receptor binding with radiolabeled 125I-a-bungarotoxin, which is a potent antagonist of a7 neuronal nAChRs.[8] All three peptides functioned as competitive antagonists of neuronal nAChRs in a dose-dependent manner (Figure S2). CDX displayed a Ki value of 0.187 mm, which is approximately 20– 40 times lower than those of Pocket_CDX and Cyclo_CDX (Table 1). Not surprisingly, CDX is substantially less potent than candoxin in nAChRs binding. The difference in potency is likely attributable, at least in part, to a loss of entropy for CDX, as it is unstructured in aqueous solution, as indicated by circular dichroism spectroscopic analysis (Figure S3). Based on the structure of candoxin (PDB code: 1JGK) elucidated by NMR spectroscopy, and the crystal structure of acetylcholine-binding protein (AChBP; PDB code: 1YI5) that shares a high degree of sequence identity with the extracellular ligand-binding domain of a7 neuronal nAChRs, we conducted molecular modeling and docking studies to better understand the interactions of the a7 nAChR with the CDX peptides. All three peptides adopt hairpinlike conformations that feature a unique electrostatic potential with segregating cationic and anionic patches (Figure S5,S6). The highly charged tip of CDX penetrates deeply into the hydrophobic binding pocket formed by two adjacent subunits of the a7 nAChR, and the binding is dominated by cation–p Table 1: Experimentally determined versus predicted free energies of binding of all synthetic peptides with a7 neuronal nAChR (DG=1.3636logKi ). Peptide Ki [mm] Experimental DG [kcalmol1 ] Estimated DG [kcalmol1 ] CDX 0.187 9.17 9.59 Pocket_CDX 4.60 7.28 8.17 [*] Dr. C. Zhan, Cyclo_CDX 7.11 7.02 7.85 [+] B. Li,[+] Dr. X. Wei, Dr. W. Fu, Prof. W. Lu School of Pharmacy & Key Laboratory of Smart Drug Delivery Ministry of Education & PLA, Fudan University Shanghai 201203 (P.R. China) Fax: (+86) 21-5198-0090 E-mail: weifuuh@gmail.com wylu@shmu.edu.cn Dr. L. Hu, Prof. L. Feng State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai 201203 (P.R. China) [ + ] These authors contributed equally to this work. [**] This work was supported by National Basic Research Program of China (973 Program) 2007CB935800 and 2010CB934000, the “Key New Drug Creation Program” 2009ZX09310-006, and Shanghai Nanotechnology Program (0953nm03300). Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201100875. Angew. Chem. Int. Ed. 2011, 50, 1 – 5 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 These are not the final page numbers!
Communications consistent with the cytochemical staining and com- petitive binding data. To examine the efficiency of CDX in facilitating nAChR Y194 mediated drug delivery to the brain, we functionalized oopC polymeric micelle-based delivery vehicle made up of biod gradable poly(ethylene glycol)-poly (D, L-lactide)(PEG-PLA) with CDX. The PEg-PLa micelle. which is a self-assembled EIS nanoparticulate carrier that features a hydrophilic shell and a high-capacity, cargo-loading hydrophobic core, is widely used 167 for the systemic delivery of various hydrophobic drugs and imaging probes. I The CDX-PEG-PLA micelle is spherical oop with a mean diameter of 39 nm, as analyzed by dynamic light CDX scattering and atomic force microscopic techniques (Fig- ure S10). For comparative pharmacokinetic studies, we teractions of CDX with the a7 nAChR. The injected normal KM mice with CDX-decorated and unmodi the receptor is shown in blue and the fied micelles that encapsulate the fluorescent dye coumarin 6, subunit in yellow. The functionally important loop and chromatographically measured the time-dependent cou and loop F in orange. CDX is depicted as a gr en ormlike structure. Residues involved in binding are represented by marin concentrations in the blood and brain. while function- sticks, and hydrogen-bond networks are denoted by black dashed alization of micelles by CDX had little impact on the pharmacokinetics of coumarin in the blood, CDX decoration of micelles significantly improved the bioavailability of coumarin in the brain as evidenced by an increase of and electrostatic interactions(Figure 1). Similar modes of in AUC (area under the curve)(Table S1). These in vivo data binding were observed with Pocket_CDX and Cyclo_CDx suggest that CDX is capable of enhancing drug transport to (Figure S7), although truncation or cyclization of CDx the CNs necessarily altered critical subsite interactions seen with To further evaluate the potential therapeutic value of CDX and contributed to a pronounced decrease in binding CDX-conjugated drug delivery systems, we studied therapeu affinity for the receptor. The contact residues identified by tic efficacy of paclitaxel-loaded CDX-PEG-PLA micelles in our docking simulation were experimentally demonstrated to a xenograft mouse model of human glioblastoma multiforme be important for the binding of three-finger toxins to the a7 (GBM). We intravenously injected three randomly divided nAChR or its homologues 9 Importantly, the binding affin- groups of nude mice bearing intracranial U87 glioblastoma ities of all three peptides calculated for the a7 nAChR by X-(n=6) with paclitaxel-encapsulated CDX-PEG-PLA good agreement with the tally micelles, drug-loaded, unmodified mPEG-PLA micelles, determined K; values(Table 1), thus computationally validat- and saline, respectively. As shown in Figure 3, in the absence ing the design of these candoxin-derived peptides as func- of CDX, treatment by paclitaxel at a dose of 10 mg per kg of tional antagonists of the a7 nAchR body weight(at 5, 10, and 15 days post-tumor implantation) To demonstrate the interaction of CDX with the neuronal did little in improving mouse survival, registering a median nAChR receptor in vitro, we incubated biotinylated CDX survival of 20 days versus 19 days for the untreated(saline with primary rat brain capillary endothelial cells, followed by group. In contrast, in the presence of CDX, paclitaxel fluorescent staining with fluorescein isothiocyanate(FITC) treatment significantly prolonged the average survival time labeled avidin. An equal molar concentration of biotin was to 27 days(p<0.01, log-rank analysis). In a separate in viv used as a negative control. The fluorescence microscopic images shown in Figure 2 clearly exhibit a specific binding of the cell surface receptors by CDX; this binding is entirely DAPI FITC m PEG-PLA-PIX Figure 3. Kaplan-Meier survival curves of nude bearing intra- Figure 2. Fluorescently stained CDX bound to primary rat brain 15 days post glioblastoma implantation) of CDX-PEG-PLA-PTX pillary endothelial cells. 10 HM of biotinylated CDX (A)or biotin(B) micelles significantly longer than the control groups tha was incubated with fixed cells at 4C overnight before adding FITc. received mPEG-PLA-PTX micelles or physiological saline(P< 0.01 sualization. Scale bars: 50 um. 2o11 Wiley-VCH Verlag GmbH& Co KGaA, Weinheim Angew. Chem. Int. Ed. 2011, 50, 1-5 Ck These are not the final page numbers!
and electrostatic interactions (Figure 1). Similar modes of binding were observed with Pocket_CDX and Cyclo_CDX (Figure S7), although truncation or cyclization of CDX necessarily altered critical subsite interactions seen with CDX and contributed to a pronounced decrease in binding affinity for the receptor. The contact residues identified by our docking simulation were experimentally demonstrated to be important for the binding of three-finger toxins to the a7 nAChR or its homologues.[9] Importantly, the binding affinities of all three peptides calculated for the a7 nAChR by XScore[10] are in good agreement with the experimentally determined Ki values (Table 1), thus computationally validating the design of these candoxin-derived peptides as functional antagonists of the a7 nAChR. To demonstrate the interaction of CDX with the neuronal nAChR receptor in vitro, we incubated biotinylated CDX with primary rat brain capillary endothelial cells, followed by fluorescent staining with fluorescein isothiocyanate (FITC) labeled avidin. An equal molar concentration of biotin was used as a negative control. The fluorescence microscopic images shown in Figure 2 clearly exhibit a specific binding of the cell surface receptors by CDX; this binding is entirely consistent with the immunocytochemical staining and competitive binding data. To examine the efficiency of CDX in facilitating nAChRmediated drug delivery to the brain, we functionalized a polymeric micelle-based delivery vehicle made up of biodegradable poly(ethylene glycol)-poly(d,l-lactide) (PEG–PLA) with CDX. The PEG–PLA micelle, which is a self-assembled nanoparticulate carrier that features a hydrophilic shell and a high-capacity, cargo-loading hydrophobic core, is widely used for the systemic delivery of various hydrophobic drugs and imaging probes.[11] The CDX–PEG–PLA micelle is spherical with a mean diameter of 39 nm, as analyzed by dynamic light scattering and atomic force microscopic techniques (Figure S10). For comparative pharmacokinetic studies, we injected normal KM mice with CDX-decorated and unmodified micelles that encapsulate the fluorescent dye coumarin 6, and chromatographically measured the time-dependent coumarin concentrations in the blood and brain. While functionalization of micelles by CDX had little impact on the pharmacokinetics of coumarin in the blood, CDX decoration of micelles significantly improved the bioavailability of coumarin in the brain as evidenced by an increase of 100% in AUC (area under the curve) (Table S1). These in vivo data suggest that CDX is capable of enhancing drug transport to the CNS. To further evaluate the potential therapeutic value of CDX-conjugated drug delivery systems, we studied therapeutic efficacy of paclitaxel-loaded CDX–PEG–PLA micelles in a xenograft mouse model of human glioblastoma multiforme (GBM). We intravenously injected three randomly divided groups of nude mice bearing intracranial U87 glioblastoma (n = 6) with paclitaxel-encapsulated CDX–PEG–PLA micelles, drug-loaded, unmodified mPEG–PLA micelles, and saline, respectively. As shown in Figure 3, in the absence of CDX, treatment by paclitaxel at a dose of 10 mg per kg of body weight (at 5, 10, and 15 days post-tumor implantation) did little in improving mouse survival, registering a median survival of 20 days versus 19 days for the untreated (saline) group. In contrast, in the presence of CDX, paclitaxel treatment significantly prolonged the average survival time to 27 days (p < 0.01, log-rank analysis). In a separate in vivo Figure 1. Modeled interactions of CDX with the a7 nAChR. The principal (+) subunit of the receptor is shown in blue and the complementary () subunit in yellow. The functionally important loop C is shown in purple and loop F in orange. CDX is depicted as a green wormlike structure. Residues involved in binding are represented by sticks, and hydrogen-bond networks are denoted by black dashed lines. Figure 2. Fluorescently stained CDX bound to primary rat brain capillary endothelial cells. 10 mm of biotinylated CDX (A) or biotin (B) was incubated with fixed cells at 48C overnight before adding FITCconjugated avidin for visualization. Scale bars: 50 mm. Figure 3. Kaplan–Meier survival curves of nude mice bearing intracranial U87 glioblastoma. Mice that received three doses (at 5, 10 and 15 days post glioblastoma implantation) of CDX–PEG–PLA–PTX micelles survive significantly longer than the control groups that received mPEG–PLA–PTX micelles or physiological saline (P<0.01, log-rank analysis). Communications 2 www.angewandte.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2011, 50, 1–5 These are not the final page numbers!
Angewandte study, we treated two groups of U87 tumor-bearing nude mice tation, the dye accumulated to the site of tumor(Fig- (n=10)using a different regimen-a lower dose of paclitaxel ure 4B)a phenomenon likely attributable to enhanced at 6 mgkg injected at 4, 8, 12, and 16 days post-tumor permeability and retention(EPR)effects seen with nano- implantation. The group that received CDX-decorated, drug- particles and solid tumors at advanced stages. Tumor loaded micelles survived an average of 25.5 days(Figure S11), progression-dependent EPR effects may also contribute to which is significantly longer than the median survival of the marked difference in survival between treatment groups 21.5 days for the untreated(saline)group(p<0.01, log-rank with and without CDX, despite a CDX-instigated, modest analysis). Collectively, these survival data suggest that CDX, improvement in bioavailability as measured in normal mice when conjugated to a delivery vehicle, can enhance the GBM is the most common primary malignant brain tumor nerapeutic efficacy of traditional chemotherapy drugs for where the BBB impedes effective chemotherapy and results brain tumors in an extremely poor prognosis 3 Drug delivery systems that nAChRs-mediated, improved drug transport to the brain efficiently cross the BBB are urgently needed for the is likely responsible for the enhanced therapeutic efficacy of treatment of GBM. We have described CDX, which is a paclitaxel in the treatment of glioblastoma. To better under- candoxin-derived peptide ligand of neuronal nAChRs, as a stand the mechanisms by which CDX-PEG-PLA micelles novel targeting molecule that enables efficient drug delivery work in vivo, we analyzed the biodistribution of CDX-Peg- to the brain by overcoming the BBB. In vitro and in vivo PLA and mPEG-PLA micelles in intracranial U87 glioblas- studies demonstrate the brain-targeting efficiency of CDX toma-bearing nude mice by using an encapsulated near- and validate its potential value in improving therapeutic infrared fluorescent dye DiR as an indicator. As shown in efficacy of existing anticancer drugs such as paclitaxel for the Figure 4 A, caudal vein injection of unmodified mPEG-PLA treatment of GBM. As nAChRs are widely expressed in the micelles at 5 days post-glioblastoma implantation resulted in brain, CDX-inspired, micelle-based drug delivery systems may be broadly applicable to therapeutic intervention for other forms of cns diseases as well Experimental Section ompetitive binding assays: The binding affinities of all peptides with euronal nAChRs were determined by a radiolabeled competition assay following reported protocols. A more detailed description of the assay conditions is given in the Supporting Information. Immunofluorescence: After fixing by an iced methanol/acetone solution(1: 1, v/v) for 5 min and permeation by 0. 2% Triton X-100 primary rat brain capillary endothelial cells were incubated with biotinylated CDX (10 HM) overnight at 4C, and then with FITc- avidin(0.5 mgmL-; dissolved in phosphate-buffered saline(PBS) containing 1% bovine serum albumen(BSA)) for 30 min at room temperature. After that, cells were washed with PBS and examined by fluorescence microscopy(IX51, Olympus, Japan) Biodistribution of CDX-PEG-PLA micelles: To study the biodistribution of CDX-PEG-PLA micelles, DIR-encapsulated mPEG-PLA or CDX-PEG-PLA micelles(100 uL) were injecte Figure 4. Near-infrared optical images of DiR encapsulated micelles into the tail vein of intracranial glioblastoma bearing nude mice at 5 injected in the caudal vein of intracranial glioblastoma-bearing nude and 15 days post-tumor implantation. The near infrared imaging was mice at 5 days(A)and 15 days(B)post-tumor implantation. The mice conducted using an in vivo imaging system(FX Pro, Kodak, USA)at on the left hand side in four different time points after injection. injected with DiR-encapsulated mPEG-PLA micelles, and the mice on the right-hand side were injected with CDX-PEG-PLA micelles loaded Received: February 3, 2011 Revised: March 31. 2011 after injection of DiR- encapsulated CDX-PEG-PLA micelles, and the Published online:■■■■,■■■■ drug delivery micelles nanomedicine. peptid no accumulation of Dir in the brain over a period of 4 days (2 h, 12 h, 2 days, 4 days). In contrast, pronounced dye accumulation in the brain at each time point was evident with injection of CDX-PEG-PLA micelles.These data [1 a)KK. Maiti, O Y Jeon, W.S. Lee, D. C. Kim, K T Kim, T. indicate that while unmodified mPEG-PLA micelles failed Takeuchi, S Futaki, S K Chung, Angew. Chem. 2006, 118, 2973 to cross the BBB, CDX functionalization helped breach the 2978; Angew.Chem.ltEd.206.45,2907-2912;b)E. hutchinson. Nat. Rev. Neurosci. 2010.11.789. barrier and facilitated DiR transport to the brain. Interest- [2] a). M. Pardridge, Mol. Interventions 2003, 3, 90-105, ingly, tumor progression appears to exacerbate the disinte- b)B J. Spencer, I M. Verma, Proc. Natl. Acad. Sci. USA gration of the BBB. When DiR-encapsulated CDX-PEG- 104, 7594-7599: c)NJ. Abbott, L. Ronnback, E Hansson, Ne PLA micelles were introduced at 15 days post-tumor implan Rev. Neurosci. 2006. 7, 41-53: d)P. Kumar, H. Wu, J.L Chem. Int. Ed. 2o11, 5o, 1-5 2011 Wiley-VCH Verlag GmbH Co KGaA, Weinheim These are not the final page numbers! ax
study, we treated two groups of U87 tumor-bearing nude mice (n = 10) using a different regimen—a lower dose of paclitaxel at 6 mg kg1 injected at 4, 8, 12, and 16 days post-tumor implantation. The group that received CDX-decorated, drugloaded micelles survived an average of 25.5 days (Figure S11), which is significantly longer than the median survival of 21.5 days for the untreated (saline) group (p < 0.01, log-rank analysis). Collectively, these survival data suggest that CDX, when conjugated to a delivery vehicle, can enhance the therapeutic efficacy of traditional chemotherapy drugs for brain tumors. nAChRs-mediated, improved drug transport to the brain is likely responsible for the enhanced therapeutic efficacy of paclitaxel in the treatment of glioblastoma. To better understand the mechanisms by which CDX–PEG–PLA micelles work in vivo, we analyzed the biodistribution of CDX–PEG– PLA and mPEG–PLA micelles in intracranial U87 glioblastoma-bearing nude mice by using an encapsulated nearinfrared fluorescent dye DiR as an indicator. As shown in Figure 4A, caudal vein injection of unmodified mPEG–PLA micelles at 5 days post-glioblastoma implantation resulted in no accumulation of DiR in the brain over a period of 4 days (2 h, 12 h, 2 days, 4 days). In contrast, pronounced dye accumulation in the brain at each time point was evident with injection of CDX-PEG-PLA micelles. These data indicate that while unmodified mPEG-PLA micelles failed to cross the BBB, CDX functionalization helped breach the barrier and facilitated DiR transport to the brain. Interestingly, tumor progression appears to exacerbate the disintegration of the BBB. When DiR-encapsulated CDX–PEG– PLA micelles were introduced at 15 days post-tumor implantation, the dye accumulated to the site of tumor (Figure 4 B)—a phenomenon likely attributable to enhanced permeability and retention (EPR) effects seen with nanoparticles and solid tumors at advanced stages.[12] Tumorprogression-dependent EPR effects may also contribute to the marked difference in survival between treatment groups with and without CDX, despite a CDX-instigated, modest improvement in bioavailability as measured in normal mice. GBM is the most common primary malignant brain tumor where the BBB impedes effective chemotherapy and results in an extremely poor prognosis.[13] Drug delivery systems that efficiently cross the BBB are urgently needed for the treatment of GBM. We have described CDX, which is a candoxin-derived peptide ligand of neuronal nAChRs, as a novel targeting molecule that enables efficient drug delivery to the brain by overcoming the BBB. In vitro and in vivo studies demonstrate the brain-targeting efficiency of CDX and validate its potential value in improving therapeutic efficacy of existing anticancer drugs such as paclitaxel for the treatment of GBM. As nAChRs are widely expressed in the brain, CDX-inspired, micelle-based drug delivery systems may be broadly applicable to therapeutic intervention for other forms of CNS diseases as well. Experimental Section Competitive binding assays: The binding affinities of all peptides with neuronal nAChRs were determined by a radiolabeled competition assay following reported protocols.[4a] A more detailed description of the assay conditions is given in the Supporting Information. Immunofluorescence: After fixing by an iced methanol/acetone solution (1:1, v/v) for 5 min and permeation by 0.2% Triton X-100, primary rat brain capillary endothelial cells were incubated with biotinylated CDX (10 mm) overnight at 48C, and then with FITCavidin (0.5 mgmL1 ; dissolved in phosphate-buffered saline (PBS) containing 1% bovine serum albumen (BSA)) for 30 min at room temperature. After that, cells were washed with PBS and examined by fluorescence microscopy (IX51, Olympus, Japan). Biodistribution of CDX–PEG–PLA micelles: To study the biodistribution of CDX–PEG–PLA micelles, DIR-encapsulated mPEG–PLA or CDX–PEG–PLA micelles (100 mL) were injected into the tail vein of intracranial glioblastoma bearing nude mice at 5 and 15 days post-tumor implantation. The near infrared imaging was conducted using an in vivo imaging system (FX Pro, Kodak, USA) at different time points after injection. Received: February 3, 2011 Revised: March 31, 2011 Published online: && &&, &&&& .Keywords: drug delivery · micelles · nanomedicine · peptides · receptors [1] a) K. K. Maiti, O. Y. Jeon, W. S. Lee, D. C. Kim, K. T. Kim, T. Takeuchi, S. Futaki, S. K. Chung, Angew. Chem. 2006, 118, 2973 – 2978; Angew. Chem. Int. Ed. 2006, 45, 2907 – 2912; b) E. Hutchinson, Nat. Rev. Neurosci. 2010, 11, 789. [2] a) W. M. Pardridge, Mol. Interventions 2003, 3, 90 – 105, 151; b) B. J. Spencer, I. M. Verma, Proc. Natl. Acad. Sci. USA 2007, 104, 7594 – 7599; c) N. J. Abbott, L. Ronnback, E. Hansson, Nat. Rev. Neurosci. 2006, 7, 41 – 53; d) P. Kumar, H. Wu, J. L. Figure 4. Near-infrared optical images of DiR encapsulated micelles injected in the caudal vein of intracranial glioblastoma-bearing nude mice at 5 days (A) and 15 days (B) post-tumor implantation. The mice on the left-hand side in four pairwise comparisons in panel (A) were injected with DiR-encapsulated mPEG–PLA micelles, and the mice on the right-hand side were injected with CDX–PEG–PLA micelles loaded with the same dose of DiR. The mouse in panel B was sacrificed 12 h after injection of DiR-encapsulated CDX–PEG–PLA micelles, and the brain dissected for imaging. Angew. Chem. Int. Ed. 2011, 50, 1 – 5 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 3 These are not the final page numbers!
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McBride, K. E. Jung, M. H. Kim, B. L. Davidson, S. K. Lee, P. Shankar, N. Manjunath, Nature 2007, 448, 39 – 43. [3] a) J. M. Lindstrom, Ann. N. Y. Acad. Sci. 2003, 998, 41 – 52; b) C. Gotti, F. Clementi, Prog. Neurobiol. 2004, 74, 363 – 396; c) R. C. Hogg, M. Raggenbass, D. Bertrand, Rev. Physiol. Biochem. Pharmacol. 2003, 147, 1 – 46; d) T. J. Abbruscato, S. P. Lopez, K. S. Mark, B. T. Hawkins, T. P. Davis, J. Pharm. Sci. 2002, 91, 2525 – 2538. [4] a) C. Zhan, Z. Yan, C. Xie, W. Lu, Mol. Pharm. 2010, 7, 1940 – 1947; b) Y. Liu, R. Huang, L. Han, W. Ke, K. Shao, L. Ye, J. Lou, C. Jiang, Biomaterials 2009, 30, 4195 – 4202. [5] a) D. Tsernoglou, G. A. Petsko, R. A. Hudson, Mol. Pharmacol. 1978, 14, 710 – 716; b) V. J. Basus, G. Song, E. Hawrot, Biochemistry 1993, 32, 12290 – 12298; c) X. Lou, Q. Liu, X. Tu, J. Wang, M. Teng, L. Niu, D. J. Schuller, Q. Huang, Q. Hao, J. Biol. Chem. 2004, 279, 39094 – 39104; d) G. Polz-Tejera, J. Schmidt, H. J. Karten, Nature 1975, 258, 349 – 351. [6] a) A. Roy, X. Zhou, M. Z. Chong, D. DHoedt, C. S. Foo, N. Rajagopalan, S. Nirthanan, D. Bertrand, J. Sivaraman, R. M. Kini, J. Biol. Chem. 2010, 285, 8302 – 8315; b) R. E. Hibbs, G. Sulzenbacher, J. Shi, T. T. Talley, S. Conrod, W. R. Kem, P. Taylor, P. Marchot, Y. Bourne, EMBO J. 2009, 28, 3040 – 3051; c) T. L. Lentz, P. T. Wilson, E. Hawrot, D. W. Speicher, Science 1984, 226, 847 – 848; d) T. L. Lentz, E. Hawrot, P. T. Wilson, Proteins Struct. Funct. Genet. 1987, 2, 298 – 307. [7] a) S. Nirthanan, E. Charpantier, P. Gopalakrishnakone, M. C. Gwee, H. E. Khoo, L. S. Cheah, D. Bertrand, R. M. Kini, J. Biol. Chem. 2002, 277, 17811 – 17820; b) S. Nirthanan, E. Charpantier, P. Gopalakrishnakone, M. C. Gwee, H. E. Khoo, L. S. Cheah, R. M. Kini, D. Bertrand, Br. J. Pharmacol. 2003, 139, 832 – 844. [8] a) I. W. Jones, J. Barik, M. J. ONeill, S. Wonnacott, J. Neurosci. Methods 2004, 134, 65 – 74; b) J. L. Eisel, S. Bertrand, J. L. Galzi, A. Devillers-Thiery, J. P. Changeux, D. Bertrand, Nature 1993, 366, 479 – 483. [9] a) K. Brejc, W. J. van Dijk, R. V. Klaassen, M. Schuurmans, J. van Der Oost, A. B. Smit, T. K. Sixma, Nature 2001, 411, 269 – 276; b) Y. Bourne, T. T. Talley, S. B. Hansen, P. Taylor, P. Marchot, EMBO J. 2005, 24, 1512 – 1522; c) P. H. Celie, I. E. Kasheverov, D. Y. Mordvintsev, R. C. Hogg, P. van Nierop, R. van Elk, S. E. van Rossum-Fikkert, M. N. Zhmak, D. Bertrand, V. Tsetlin, T. K. Sixma, A. B. Smit, Nat. Struct. Mol. Biol. 2005, 12, 582 – 588. [10] R. Wang, L. Lai, S. Wang, J. Comput.-Aided Mol. Des. 2002, 16, 11 – 26. [11] a) K. Yasugi, Y. Nagasaki, M. Kato, K. Kataoka, J. Controlled Release 1999, 62, 89 – 100; b) N. Nasongkla, E. Bey, J. Ren, H. Ai, C. Khemtong, J. S. Guthi, S. F. Chin, A. D. Sherry, D. A. Boothman, J. Gao, Nano Lett. 2006, 6, 2427 – 2430; c) N. Kang, M. E. Perron, R. E. Prudhomme, Y. Zhang, G. Gaucher, J. C. Leroux, Nano Lett. 2005, 5, 315 – 319. [12] a) J. J. Verhoeff, O. van Tellingen, A. Claes, L. J. Stalpers, M. E. van Linde, D. J. Richel, W. P. Leenders, W. R. van Furth, BMC Cancer 2009, 9, 444; b) J. R. Ewing, S. L. Brown, M. Lu, S. Panda, G. Ding, R. A. Knight, Y. Cao, Q. Jiang, T. N. Nagaraja, J. L. Churchman, J. D. Fenstermacher, J. Cereb. Blood Flow Metab. 2006, 26, 310 – 320; c) L. L. Muldoon, C. Soussain, K. Jahnke, C. Johanson, T. Siegal, Q. R. Smith, W. A. Hall, K. Hynynen, P. D. Senter, D. M. Peereboom, E. A. Neuwelt, J. Clin. Oncol. 2007, 25, 2295 – 2305. [13] a) C. Zhan, B. Gu, C. Xie, J. Li, Y. Liu, W. Lu, J. Controlled Release 2010, 143, 136 – 142; b) V. Laquintana, A. Trapani, N. Denora, F. Wang, J. M. Gallo, G. Trapani, Expert Opin. Drug Delivery 2009, 6, 1017 – 1032; c) F. I. Staquicini, M. G. Ozawa, C. A. Moya, W. H. Driessen, E. M. Barbu, H. Nishimori, S. Soghomonyan, L. G. Flores 2nd, X. Liang, V. Paolillo, M. M. Alauddin, J. P. Basilion, F. B. Furnari, O. Bogler, F. F. Lang, K. D. Aldape, G. N. Fuller, M. Hook, J. G. Gelovani, R. L. Sidman, W. K. Cavenee, R. Pasqualini, W. Arap, J. Clin. Invest. 2011, 121, 161 – 173. Communications 4 www.angewandte.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2011, 50, 1–5 These are not the final page numbers!
aNgewandte Communications Nanomedicine Principal Candid candoxin: A 16-residue peptide Ligand binding CDX)that is derived from candoxin C. Zhan, B. Li, L Hu, X. Wei, L. Feng, binds with a high affinity to nicotinic W. Fu s W. Lux Candoxin which are highly expressed on the Micelle-Based Brain-Targeted Drug brain barrier In vivo biodistribution and Delivery Enabled by a Nicotine omplementary he anti-glioblastoma effect indicate the Acetylcholine Receptor Ligand Subunit (- Chem. Int. Ed. 2o11, 5o, 1-5 2011 Wiley-VCH Verlag GmbH& Co KGaA, Weinheim These are not the final page numbers ax
Communications Nanomedicine C. Zhan, B. Li, L. Hu, X. Wei, L. Feng, W. Fu,* W. Lu* &&&& —&&&& Micelle-Based Brain-Targeted Drug Delivery Enabled by a Nicotine Acetylcholine Receptor Ligand Candid candoxin: A 16-residue peptide (CDX) that is derived from candoxin binds with a high affinity to nicotinic acetylcholine receptors (see picture), which are highly expressed on the blood– brain barrier. In vivo biodistribution and the anti-glioblastoma effect indicate the potential of CDX as a ligand to enable brain-targeted drug delivery. Angew. Chem. Int. Ed. 2011, 50, 1 – 5 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 5 These are not the final page numbers!