2合5EAB www.Materialsviews.com 3 Choline-Derivate-Modified Nanoparticles for E Brain-Targeting Gene Dellvery Jianfeng Li, Lu Zhou, Deyong Ye, Shixian Huang, Kun Shao, Rongqin Huang, E Liang Han, Yang Liu, Shuhuan Liu, Liya Ye, Jinning Lou, and Chen Jiang The major obstacle for drug delivery to the central nervous system BBB choline transporter( BBB ChT) could be used to deliver D, L- (CNS)is the blood-brain barrier(BBB), which protects the CNS 2-amino-7-bis[(2-chloroethyl)amino]-1, 2, 3, 4-tetrahydro-2-naph from potentially harmful xenobiotics and endogenous molecules thoic acid (D, L- NAM), ketoprofen, and N-n-octylnicotinium iodide to ensure an optimal environment for brain function. I Despite (NONI), respectively. /. Despite these successful applications, this natural barricade, small molecules and macromolecules the CMT mechanism remains an untapped resource for delivery including peptides and proteins could be transported into the systems. In this work, CMT mechanism was challenged to facili- CNS to maintain its normal physiological function via the endog. tate the brain-targeting drug-delivery system across the BBB enous BBB transporters. There are three families of endogenous Choline is a biochemical precursor of the neurotransmitter BBB transporters: carrier-mediated transporters (CMT), active acetylcholine and other essential components of cell membrane efflux transporters (AET), and receptor-mediated transporters phospholipids such as phosphatidylcholine. The high choline Mr). The CMT and AET systems are mainly responsible for concentration in the brain demonstrates the amazing ability of the the transport of small molecules, while the RMT systems are BBB ChT to transport choline across the BBB 9.10 Furthermore, onsible for endogenous large molecules. 2.3 the low physiologic plasma concentration of choline(approxi- Drugs or drug-delivery systems can be modified with the sub- mately 25% of the Michaelis-Menten constant, Km) makes the strates of these transporters to realize their accumulation in the BBB ChT free to transport choline derivates without interrupting CNS. The RMT mechanism has been mainly investigated and the supply of CNS choline I1 Therefore, BBB ChT is considered utilized for brain-targeting drug delivery. 2 For example, the lig. a potential target for active drug delivery into the CNS ands of transferrin receptor and insulin receptor have been used A quaternary ammonium group and a free hydroxyl to modify polymers for constructing brain-targeting drug-delivery group were suggested as the key requirements for BBB ChT stems to transport small molecules, proteins, and gene drugs substrates IS As a native BBB ChT substrate, choline was no into the CNS 4.1 A number of specific transporters are expressed suitable for further modification. Recent studies revealed that on the brain capillary endothelial cells(BCECs), which are bis-quaternary ammonium compounds could also be applied as sponsible for the endogenous and exogenous nutrient supplies BBB ChT substrates with even higher affinity. 12, 13) Thus, bis- for the brain. They possess inspiring features including high quaternary ammonium compounds were challenged to modify ansport capacity, selectivity, and an adequate transfer rate. 6 dendrimers for constructing the brain-targeting drug-delivery Thus, a native BBB nutrient transporter could be utilized as an system without affecting their BBB Cht affinity. alternative strategy to enable polar, water-soluble drugs to cross First, a series of novel bis-quaternary ammonium compounds the BBB via a CMT mechanism. Several successful applications with high BBB ChT affinity were designed. There were two major have been reported. For example, the BBB large neutral amino factors affecting the BBB ChT affinity of these compound acid transport system, the glucose transporter(GluTl), and the i) the lipophilicity of the quaternary ammonium moieties and ii) the length and conformation rigidity of the linkers between two quaternary ammonium moieties. I) Given these factors, high J. F. Li, S.X. Huang, K. Shao, R.Q. Huang, L Han, Y Liu, S.H. Liu, lipophilic isoquinoline(4a)and relatively low lipophilic 3-methyl- Prof. C. Jiang pyridine(4b) were chosen as quaternary ammonium moieties Key Laboratory of Smart Drug Delive try of Education and PLA Department of Pharmaceutics (,5-bis(3-bromopropoxy)phenyl)methanol(3a, 11 carbons) and 3, 5-bis(4-bromobutoxy)phenyl)methanol(3b, 13 carbons)were Fudan Universi elected as linkers(Scheme 1). High conformation rigidity of the 26 Zhangheng Road, Shanghai 201203, China benzene ring could result in higher BBB ChT affinity and benzylic E-mail: jiangchen @sh hydroxyl could also provide a free reactive site for conjugation to a Dr L. Zhou. Prof D. Y. Ye brain-targeting drug-delivery system Based on the above consider- Key Laboratory of Smart Drug Delive Ministry of Education and PLA Department of Medical Chemistry ations, four bis-quaternary ammonium compounds (5, 6, 7, 8)have been crossover designed, synthesized (as shown in Scheme 1) Fudan Universi and characterized by mass spectrometry(MS), ' H NMR, and 1C 26 Zhangheng Road, Shanghai 201203, China NMR (see Supporting Information). The characterization showed successful synthesis of compounds 5, 6, 7, and 8 Institute of Clinical Medical Sciences To verify the design strategy, the BBB ChT affinityiesof the syn thesized bis-quaternary ammonium compounds were compared by evaluation of their ability to inhibit the [Hl-choline chloride Do:10.1002/adma201101899 uptake by BCECs(Figure la). The ICso value(concentration 4516wileyonlinelibrary.com 9 2011 WILEY-VCH Verlag GmbH Co KGaA, Weinheim Ad. Mater2011,23,4516-4520
4516 © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advmat.de www.MaterialsViews.com COMMUNICATION wileyonlinelibrary.com Adv. Mater. 2011, 23, 4516–4520 J. F. Li , S. X. Huang , K. Shao , R. Q. Huang , L. Han , Y. Liu , S. H. Liu , Prof. C. Jiang Key Laboratory of Smart Drug Delivery Ministry of Education and PLA Department of Pharmaceutics School of Pharmacy Fudan University 826 Zhangheng Road, Shanghai 201203, China E-mail: jiangchen@shmu.edu.cn Dr. L. Zhou , Prof. D. Y. Ye Key Laboratory of Smart Drug Delivery Ministry of Education and PLA Department of Medical Chemistry School of Pharmacy Fudan University 826 Zhangheng Road, Shanghai 201203, China L. Y. Ye , Prof. J. N. Lou Institute of Clinical Medical Sciences China–Japan Friendship Hospital The Ministry of Health, Beijing, China DOI: 10.1002/adma.201101899 The major obstacle for drug delivery to the central nervous system (CNS) is the blood–brain barrier (BBB), which protects the CNS from potentially harmful xenobiotics and endogenous molecules to ensure an optimal environment for brain function. [1] Despite this natural barricade, small molecules and macromolecules including peptides and proteins could be transported into the CNS to maintain its normal physiological function via the endogenous BBB transporters. There are three families of endogenous BBB transporters: carrier-mediated transporters (CMT), active effl ux transporters (AET), and receptor-mediated transporters (RMT). The CMT and AET systems are mainly responsible for the transport of small molecules, while the RMT systems are responsible for endogenous large molecules. [2,3] Drugs or drug-delivery systems can be modifi ed with the substrates of these transporters to realize their accumulation in the CNS. The RMT mechanism has been mainly investigated and utilized for brain-targeting drug delivery. [2] For example, the ligands of transferrin receptor and insulin receptor have been used to modify polymers for constructing brain-targeting drug-delivery systems to transport small molecules, proteins, and gene drugs into the CNS. [4,5] A number of specifi c transporters are expressed on the brain capillary endothelial cells (BCECs), which are responsible for the endogenous and exogenous nutrient supplies for the brain. They possess inspiring features including high transport capacity, selectivity, and an adequate transfer rate. [6] Thus, a native BBB nutrient transporter could be utilized as an alternative strategy to enable polar, water-soluble drugs to cross the BBB via a CMT mechanism. Several successful applications have been reported. For example, the BBB large neutral amino acid transport system, the glucose transporter (GluT1), and the BBB choline transporter (BBB ChT) could be used to deliver D , L - 2-amino-7-bis[(2-chloroethyl)amino]-1,2,3,4-tetrahydro-2-naphthoic acid ( D , L -NAM), ketoprofen, and N -n-octylnicotinium iodide (NONI), respectively. [7,8] Despite these successful applications, the CMT mechanism remains an untapped resource for delivery systems. In this work, CMT mechanism was challenged to facilitate the brain-targeting drug-delivery system across the BBB. Choline is a biochemical precursor of the neurotransmitter acetylcholine and other essential components of cell membrane phospholipids such as phosphatidylcholine. The high choline concentration in the brain demonstrates the amazing ability of the BBB ChT to transport choline across the BBB. [9,10] Furthermore, the low physiologic plasma concentration of choline (approximately 25% of the Michaelis–Menten constant, Km ) makes the BBB ChT free to transport choline derivates without interrupting the supply of CNS choline. [11] Therefore, BBB ChT is considered a potential target for active drug delivery into the CNS. A quaternary ammonium group and a free hydroxyl group were suggested as the key requirements for BBB ChT substrates. [8] As a native BBB ChT substrate, choline was not suitable for further modifi cation. Recent studies revealed that bis-quaternary ammonium compounds could also be applied as BBB ChT substrates with even higher affi nity. [12,13] Thus, bisquaternary ammonium compounds were challenged to modify dendrimers for constructing the brain-targeting drug-delivery system without affecting their BBB ChT affi nity. First, a series of novel bis-quaternary ammonium compounds with high BBB ChT affi nity were designed. There were two major factors affecting the BBB ChT affi nity of these compounds: i) the lipophilicity of the quaternary ammonium moieties and ii) the length and conformation rigidity of the linkers between two quaternary ammonium moieties. [13] Given these factors, high lipophilic isoquinoline ( 4a ) and relatively low lipophilic 3-methylpyridine ( 4b ) were chosen as quaternary ammonium moieties. (3,5-bis(3-bromopropoxy)phenyl)methanol ( 3a , 11 carbons) and (3,5-bis(4-bromobutoxy)phenyl)methanol ( 3b , 13 carbons) were selected as linkers ( Scheme 1 ). High conformation rigidity of the benzene ring could result in higher BBB ChT affi nity and benzylic hydroxyl could also provide a free reactive site for conjugation to a brain-targeting drug-delivery system. Based on the above considerations, four bis-quaternary ammonium compounds ( 5 , 6 , 7 , 8 ) have been crossover designed, synthesized (as shown in Scheme 1 ), and characterized by mass spectrometry (MS), 1 H NMR, and 13 C NMR (see Supporting Information). The characterization showed successful synthesis of compounds 5 , 6 , 7 , and 8 . To verify the design strategy, the BBB ChT affi nityiesof the synthesized bis-quaternary ammonium compounds were compared by evaluation of their ability to inhibit the [ 3 H]-choline chloride uptake by BCECs ( Figure 1 a). The IC 50 value (concentration Jianfeng Li , Lu Zhou , Deyong Ye , Shixian Huang , Kun Shao , Rongqin Huang , Liang Han , Yang Liu , Shuhuan Liu , Liya Ye , Jinning Lou , and Chen Jiang * Choline-Derivate-Modifi ed Nanoparticles for Brain-Targeting Gene Delivery
A合 ww. MaterialsViews com a 2bm=2 3b 0、Br 风O On R 7 P2R= 8m=2R= Scheme 1. Synthesis of bis-quaternary ammonium compounds 5-8. Reagents and conditions: a)K2CO3, ACN, reflux 4 h and b)65C, 1 h compound 6 compound 7 (b) (d ∠P into BCECs Sponse relationship for the inhibition of PHI-choline chloride uptake by ake of PHI 10 nM)into BCECs was measured with a 10-min incubation in the presence of Nacl at pH 7.4 he absence(control) or presenc oncentrations of various compounds. Data are expressed as mean t standard deviation(SD ber of samples, n= 4). b)Stru relationship indicated by ICso values of various compounds. c) In vivo imaging of mice administrated with 50 uM10BODIPY(right) and of equal fluorescence(left). d) In vitro imaging of the main organs of mice administered with 50 HM10-BODIPY (low panel) and free BOD fluorescence(upper panel). Images were taken 2 h after administration. Intensity of the signal: dark red is the strongest and dark blue is as shown by the bar. Ad. Mater2011,23,4516-4520 9 2011 WILEY-VCH Verlag GmbH Co KGaA, Weinheim wileyonlinelibrary. com 4517
4517 www.advmat.de www.MaterialsViews.com © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim COMMUNICATION Adv. Mater. 2011, 23, 4516–4520 wileyonlinelibrary.com Figure 1 . a) Dose–response relationship for the inhibition of [ 3 H]-choline chloride uptake by various compounds. Uptake of [ 3 H]-choline chloride (10 n M ) into BCECs was measured with a 10-min incubation in the presence of NaCl at pH 7.4 either in the absence (control) or presence of assigned concentrations of various compounds. Data are expressed as mean ± standard deviation (S.D.) (number of samples, n = 4). b) Structure–activity relationship indicated by IC 50 values of various compounds. c) In vivo imaging of mice administrated with 50 μM10 -BODIPY (right) and free BODIPY of equal fl uorescence (left). d) In vitro imaging of the main organs of mice administered with 50 μM10 -BODIPY (low panel) and free BODIPY of equal fl uorescence (upper panel). Images were taken 2 h after administration. Intensity of the signal: dark red is the strongest and dark blue is the weakest, as shown by the bar. Scheme 1 . Synthesis of bis-quaternary ammonium compounds 5 - 8 . Reagents and conditions: a) K 2 CO 3 , ACN, refl ux 4 h and b) 65 ° C, 1 h. + 1 2a n=1 2b n=2 HO OH OH O O OH Br Br Br Br n n n 3a n=1 3b n=2 a N R b O O OH Br Br n n + OH O O N+ N R + R 4a R= 4b R= N N 5 n=1 R= 6 n=1 R= 7 n=2 R= 8 n=2 R= N N N N Br- n n Br-
2合5EAB www.advmat.de necessary for 50% inhibition) was calculated by One site-Fit 123 amino groups per molecule) have been utilized for gene logICso (an equation in GraphPad Prism 5). For compound 5, delivery to brain. [14, 151 Compound 10 was conjugated to the 6, 7, and 8 and choline chloride, the ICso values were 80 25 HM, surface of the DGL via a-malemidyl-u-N-hydroxysuccinimidy 102.9 HM,4.265 HM, 49.08 HM, and 14.50 uM, respectively. Com. polyethyleneglycol (NHS-PEG-MAL) and further constructed parison of compound 5 with 6 and 7 with 8(Figure 1B) indicated the gene delivery system. Details of the synthesis and character that compounds with isoquinoline as quaternary ammonium ization of DGL- PEG/pDNA(plasmid DNA)and DGL-PEG-10/ moieties possessed higher affinity. The cationic binding site of pDNA nanoparticles(NPs)are shown in Figure S1( Supporting E BBB ChT may locate inside a hydrophobic pocket, thus higher Information) lipophilicity of moieties could gain higher affinity. Comparison To determine whether BBB ChT could mediate the brain of compound 5 with 7 and 6 with 8( Figure 1b) suggested that accumulation of a gene-delivery system, the cellular uptake the optimal length of linker was 13 carbons. It may attribute mechanism of NPs with YOYO-1-labeled(Invitrogen, Y3601) the potential binding sites of quaternary ammonium moie. pDNa was investigated. The cellular uptake of DGL-PEG-10 ties with BBB ChT: i) one BBB ChT has several cationic binding PDNA (Figure 2b) was much more than that of DGL-PEGI sites and two quaternary ammonium cations bind to two of PDNA(Figure 2a). Excess choline chloride could inhibit the them and i) one BBB ChT has only one cationic binding site uptake of DGL- PEG-10/pDNA (Figure 2c). The uptake of DGL- and two quaternary ammonium cations bind to two nearby PEG-10/pDNA at 4oC significantly decreased( Figure 2f) BBB ChTs. Independent of the binding arrangement, the These indicated that the uptake showed energy-dependent and results verified that a distance of 13 carbons was more suit- BBB-ChT-involving character. The uptake mechanism for the compounds revealed a simple structure-activity relationship. on the BBB: i)receptor-mediated endocytosis(RME), in whis, o able. Comparison of the BBB ChT affinity of the synthesized NPs is endocytosis. There are two main kinds of endocytos The bis-quaternary ammonium compound with isoquinoline as the quaternary ammonium moieties, which were connected by a high conformation rigidity linker with 13 carbons (com- pound 7), had the highest BBB ChT affinity. Among the four (a) synthesized compounds, compound 7 had the lowest ICso value (4. 265 HM), which was even lower than that of choline chloride (14.50 HM). This indicated that compound 7 could compete efficiently with endogenous choline in binding to BBB ChT in vivo. Thus compound 7, as a novel BBB ChT substrate, was further conjugated to dendrimers for constructing the ( d) brain-targeting drug-delivery syster In order to gain more efficient conjug he benzylic hydroxyl of compoun thiol Compound 10 was the sulfhydrylation form of compound 7(Scheme Sl, Supporting Information) and its brain-targeting by labeling with a fu gent, BODIPY (4, 4-difluoro.5, 7-dimethyl-4bora-3a, 4a-diaza- 45 DGL-PEG-10/pDNA --DGL-PEG-10/pDNA+inhibitor indacene-3-propionic acid, sulfosuccinimidyl ester, sodium 3 40 salt). Compound 10BODIPY and free BODIPY of equal fluores.535 --DGL-PEG/pDNA cence were injected via the caudal vein of nude mice. As shown g 30 in Figure Ic, the fluorescence in the brain of the mice treated with compound 10-BODIPY was significantly stronger than 20 that of the mice treated with free BODIPY In vitro imaging 15 results showed that the accumulation of compound 10-BODIPY 2 10 was more hydrophilic for the two cationic moieties and led to faster excretion, thus it had more accumulation in the 0 kidneys. Traditionally it was thought that molecules with more 0102030 ipophilicity could cross the BBB more easily. It was amazing that the decreased lipophilicity of compound 10BoDIPY did not result in less, but instead more, accumulation in brain. This Figure 2. Cellular uptake of DGL-PEG/PDNA (a), DGL-PEG-10/pDNA examined by fluorescent micro- was due to the high BBB ChT affinity of compound 10. Here the scopy after a 30-min incubation at 37oC.BCECs were treated with different BBB ChT acted as a Trojan horse to increase the accumulation inhibitors including choline chloride(c), filipin complex(d), phenylarsine of compound 10-BODIPY in brain. This indicated the possibility (e). The cellular uptake of DGL-PEG-10/pDNA was also carried out at that BBB ChT could also mediate the brain accumulation of a 4C (f). Green: YOYO-1 labeled pDNA. Original magnification: x100 drug-delivery system modified with compound 10 g)Results of transport studies of NPs across the BCECs monolayer. Data is shown as Papp at assigned time points. Significance: +p 0.05; Next, compound 10 was conjugated to the drug-delivery ++p 0.01, represents DGL-PEG-10/PDNA vS. DGL-PEG-10 stem and the brain-targeting efficiency was evaluated in vitro pDNA+inhibitor, while ***p<0.001, represents DGL-PEG-10/pDNA vs and in vivo. Dendrigraft poly-L-lysines(DGLs, generation 3 with DGL-PEG/pDNA. Results are expressed as mean +S.D.(n=4) 4518wileyonlinelibrary.com 9 2011 WILEY-VCH Verlag GmbH Co KGaA, Weinheim Ad. Mater2011,23,4516-4520
4518 www.advmat.de www.MaterialsViews.com © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim COMMUNICATION wileyonlinelibrary.com Adv. Mater. 2011, 23, 4516–4520 123 amino groups per molecule) have been utilized for gene delivery to brain. [14,15] Compound 10 was conjugated to the surface of the DGL via a-malemidyl-u- N -hydroxysuccinimidyl polyethyleneglycol (NHS-PEG-MAL) and further constructed the gene delivery system. Details of the synthesis and characterization of DGL-PEG/pDNA (plasmid DNA) and DGL-PEG- 10 / pDNA nanoparticles (NPs) are shown in Figure S1 (Supporting Information). To determine whether BBB ChT could mediate the brain accumulation of a gene-delivery system, the cellular uptake mechanism of NPs with YOYO-1-labeled (Invitrogen, Y3601) pDNA was investigated. The cellular uptake of DGL-PEG- 10 / pDNA ( Figure 2 b) was much more than that of DGL-PEG/ pDNA (Figure 2 a). Excess choline chloride could inhibit the uptake of DGL-PEG- 10 /pDNA (Figure 2 c). The uptake of DGLPEG- 10 /pDNA at 4 ° C signifi cantly decreased (Figure 2 f). These indicated that the uptake showed energy-dependent and BBB-ChT-involving character. The uptake mechanism for the NPs is endocytosis. There are two main kinds of endocytosis on the BBB: i) receptor-mediated endocytosis (RME), in which a necessary for 50% inhibition) was calculated by One site-Fit logIC 50 (an equation in GraphPad Prism 5). For compound 5 , 6 , 7 , and 8 and choline chloride , the IC 50 values were 80.25 μM , 102.9 μM , 4.265 μM , 49.08 μM , and 14.50 μM , respectively. Comparison of compound 5 with 6 and 7 with 8 (Figure 1 B) indicated that compounds with isoquinoline as quaternary ammonium moieties possessed higher affi nity. The cationic binding site of BBB ChT may locate inside a hydrophobic pocket, thus higher lipophilicity of moieties could gain higher affi nity. Comparison of compound 5 with 7 and 6 with 8 (Figure 1 b) suggested that the optimal length of linker was 13 carbons. It may attribute to the potential binding sites of quaternary ammonium moieties with BBB ChT: i) one BBB ChT has several cationic binding sites and two quaternary ammonium cations bind to two of them and ii) one BBB ChT has only one cationic binding site and two quaternary ammonium cations bind to two nearby BBB ChTs. Independent of the binding arrangement, the results verifi ed that a distance of 13 carbons was more suitable. Comparison of the BBB ChT affi nity of the synthesized compounds revealed a simple structure–activity relationship. The bis-quaternary ammonium compound with isoquinoline as the quaternary ammonium moieties, which were connected by a high conformation rigidity linker with 13 carbons (compound 7 ), had the highest BBB ChT affi nity. Among the four synthesized compounds, compound 7 had the lowest IC 50 value (4.265 μM ), which was even lower than that of choline chloride (14.50 μM ). This indicated that compound 7 could compete effi ciently with endogenous choline in binding to BBB ChT in vivo. Thus compound 7 , as a novel BBB ChT substrate, was further conjugated to dendrimers for constructing the brain-targeting drug-delivery system. In order to gain more effi cient conjugation to dendrimers, the benzylic hydroxyl of compound 7 was converted to benzylic thiol. Compound 10 was the sulfhydrylation form of compound 7 (Scheme S1, Supporting Information) and its brain-targeting effi ciency was investigated in vivo by labeling with a fl uorescent agent, BODIPY (4,4-difl uoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt). Compound 10 -BODIPY and free BODIPY of equal fl uorescence were injected via the caudal vein of nude mice. As shown in Figure 1 c, the fl uorescence in the brain of the mice treated with compound 10 -BODIPY was signifi cantly stronger than that of the mice treated with free BODIPY. In vitro imaging results showed that the accumulation of compound 10 -BODIPY in the brain and kidneys increased (Figure 1 d). Compound 10 - BODIPY was more hydrophilic for the two cationic moieties and led to faster excretion, thus it had more accumulation in the kidneys. Traditionally it was thought that molecules with more lipophilicity could cross the BBB more easily. It was amazing that the decreased lipophilicity of compound 10 -BODIPY did not result in less, but instead more, accumulation in brain. This was due to the high BBB ChT affi nity of compound 10 . Here the BBB ChT acted as a Trojan horse to increase the accumulation of compound 10 -BODIPY in brain. This indicated the possibility that BBB ChT could also mediate the brain accumulation of a drug-delivery system modifi ed with compound 10 . Next, compound 10 was conjugated to the drug-delivery system and the brain-targeting effi ciency was evaluated in vitro and in vivo. Dendrigraft poly- L -lysines (DGLs, generation 3 with Figure 2 . Cellular uptake of DGL-PEG/pDNA (a), DGL-PEG- 10 /pDNA (b) with different inhibitors (c–e) were examined by fl uorescent microscopy after a 30-min incubation at 37 ° C. BCECs were treated with different inhibitors including choline chloride (c), fi lipin complex (d), phenylarsine (e). The cellular uptake of DGL-PEG- 10 /pDNA was also carried out at 4 ° C (f). Green: YOYO-1 labeled pDNA. Original magnifi cation: × 100. g) Results of transport studies of NPs across the BCECs monolayer. Data is shown as Papp at assigned time points. Signifi cance: +p < 0.05; ++p < 0.01, represents DGL-PEG- 10 /pDNA vs. DGL-PEG- 10 / pDNA + inhibitor, while ∗∗∗p < 0.001, represents DGL-PEG- 10 /pDNA vs. DGL-PEG/pDNA. Results are expressed as mean ± S.D. ( n = 4)
A合 ww. MaterialsViews com ligand-modified system binds to its cognate receptor specifically the excess choline chloride as inhibitor, Papp decreased due to to elicit cellular internalization and can be inhibited by pheny. the competition with compound 10. The incorporation of com- arsine oxide and ii) adsorptive endocytosis(AE), in which the pound 10 could lead to higher permeability across the bcecs drug-delivery system will bind to the cellular surface through monolayer. non-specific mechanisms, such as electrostatic interactions, Inspired by the in vitro results, in vivo brain-targeting effi and can be blocked by a filipin complex. Q It was reported that ciency of the NPs was investigated 2 h after injection of the Ce uptake of Angiopep (a high-affinity ligand of low-density NPs. The accumulation of ethidium monoazide bromide(EMA) poprotein-receptor-related protein) modified NPs could sig- labled pDNA in the brain treated with DGL-PEG-10/pDNA was nificantly be inhibited by phenylarsine oxide. 6 The modifica- more than that treated with DGL- PEG/pDNA(Figure 3a). Then, tion of Angiopep gave the NPs RME character. The uptake of distribution of gene expression in mouse brain was detected. NPs decreased with addition of the filipin complex(Figure 2d) For DGL-PEG/PEGFP, green fuorescent protein(GFP)expres- but not phenylarsine oxide(Figure 2e), thus showing that the sion was found mainly in fourth ventricle(Figure 3g) and main pathway of endocytosis of was AE not RME. The surface little was found in other regions( Figure 3c-f. It was reported mino groups were slightly cationic and this contributed to the that exogenous gene expression could be observed in limited AE pathway. However, unlike ordinary AE, the modification of regions around the cerebral ventricles due to their relatively compound 10 increased the capture possibility of NPs by the weak BBB. DGL-PEG/pEGFP had high GFP expression in cell membrane. Subsequently, the transport efficiency across fourth ventricle. It could be hypothesized that NPs modified the BCECs monolayer was evaluated. As shown in Figure 2g, with compound 10 were likely to accumulate in high-choline the apparent permeability (Papp) of DGL-PEG-10/pDNA was demanding regions, thus resulting in more GFP expression in much higher than that of DGL-PEG/PDNA, especially in the these regions. For DGL- PEG-10/PEGFP, GFP expression was first 15 min(DGL- PEG-10/pDNA, 26.47 cm s-: DGL-PEG/ mainly found in cortical layer(Figure 3h), caudate putamen DNA, 6.61 cm s-l). The higher permeability was probably (Figure 3i), and fourth ventricle(Figure 31). The cortical layer due to the transporting character of BBB-ChT. When adding and caudate putamen are rich in neurons and have high 10000 brain a8000 54000 三2000 DGL-PEGip GL3 DGL-PEG-10/pGL3 (d) e (g) Figure 3. a) In vivo imaging of mice administrated with DGL- PEG/pDNA (left) or DGL-PEG-10/pDNA (rigi were taken 2 h after administra. ion. b) Luciferase expression 48 h after intravenous administration of DGL- PEG/pGL3 and DGL-PEG-10 lb/c mice at a dose of 50 DNA per mouse. Luciferase expression is plotted as light units per mg protein. **p <0.01 represents DGL- GL3 VS. DGL-PEG/pGL3. Data are expressed as mean+SD(n=4). c-l) Distribution of gene expression in brains of mice treated with DGI FP(c-g)or DGL-PEG-10/PEGF (h-48 h after intravenous administration. Frozen sections(thickness of 20 um)of cortical layer(c, h), caudate putamen(d, O), hippocampus (e, i) nd substantia nigra(f, k ), and fourth ventricle (g, I)were examined by fluorescent microscopy. The sections were stained with 300 nM DAPI for 10 min at room temperature. Green: GFP Blue: cell nuclei. Original magnification: x100 Ad. Mater2011,23,4516-4520 9 2011 WILEY-VCH Verlag GmbH Co KGaA, Weinheim wileyonlinelibrary. com 4519
4519 www.advmat.de www.MaterialsViews.com © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim COMMUNICATION Adv. Mater. 2011, 23, 4516–4520 wileyonlinelibrary.com the excess choline chloride as inhibitor, Papp decreased due to the competition with compound 10 . The incorporation of compound 10 could lead to higher permeability across the BCECs monolayer. Inspired by the in vitro results, in vivo brain-targeting effi - ciency of the NPs was investigated 2 h after injection of the NPs. The accumulation of ethidium monoazide bromide (EMA) labled pDNA in the brain treated with DGL-PEG- 10 /pDNA was more than that treated with DGL-PEG/pDNA ( Figure 3 a). Then, distribution of gene expression in mouse brain was detected. For DGL-PEG/pEGFP, green fl uorescent protein (GFP) expression was found mainly in fourth ventricle (Figure 3 g) and little was found in other regions (Figure 3 c–f). It was reported that exogenous gene expression could be observed in limited regions around the cerebral ventricles due to their relatively weak BBB. [17] DGL-PEG/pEGFP had high GFP expression in fourth ventricle. It could be hypothesized that NPs modifi ed with compound 10 were likely to accumulate in high-cholinedemanding regions, thus resulting in more GFP expression in these regions. For DGL-PEG- 10 /pEGFP, GFP expression was mainly found in cortical layer (Figure 3 h), caudate putamen (Figure 3 i), and fourth ventricle (Figure 3 l). The cortical layer and caudate putamen are rich in neurons and have high ligand-modifi ed system binds to its cognate receptor specifi cally to elicit cellular internalization and can be inhibited by phenylarsine oxide and ii) adsorptive endocytosis (AE), in which the drug-delivery system will bind to the cellular surface through non-specifi c mechanisms, such as electrostatic interactions, and can be blocked by a fi lipin complex. [1] It was reported that the uptake of Angiopep (a high-affi nity ligand of low-density lipoprotein-receptor-related protein) modifi ed NPs could signifi cantly be inhibited by phenylarsine oxide. [16] The modifi cation of Angiopep gave the NPs RME character. The uptake of NPs decreased with addition of the fi lipin complex (Figure 2 d) but not phenylarsine oxide (Figure 2 e), thus showing that the main pathway of endocytosis of was AE not RME. The surface amino groups were slightly cationic and this contributed to the AE pathway. However, unlike ordinary AE, the modifi cation of compound 10 increased the capture possibility of NPs by the cell membrane. Subsequently, the transport effi ciency across the BCECs monolayer was evaluated. As shown in Figure 2 g, the apparent permeability ( Papp ) of DGL-PEG- 10 /pDNA was much higher than that of DGL-PEG/pDNA, especially in the fi rst 15 min (DGL-PEG- 10 /pDNA, 26.47 cm s − 1 ; DGL-PEG/ pDNA, 6.61 cm s − 1 ). The higher permeability was probably due to the transporting character of BBB-ChT. When adding Figure 3 . a) In vivo imaging of mice administrated with DGL-PEG/pDNA (left) or DGL-PEG- 10 /pDNA (right). Images were taken 2 h after administration. b) Luciferase expression 48 h after intravenous administration of DGL-PEG/pGL3 and DGL-PEG- 10 /pGL3 into Balb/c mice at a dose of 50 μ g DNA per mouse. Luciferase expression is plotted as light units per mg protein. ∗∗p < 0.01 represents DGL-PEG- 10 /pGL3 vs. DGL-PEG/pGL3. Data are expressed as mean ± S.D. ( n = 4). c–l) Distribution of gene expression in brains of mice treated with DGL-PEG/pEGFP (c–g) or DGL-PEG- 10 /pEGFP (h–l) 48 h after intravenous administration. Frozen sections (thickness of 20 μ m) of cortical layer (c,h), caudate putamen (d,i), hippocampus (e, j), and substantia nigra (f,k), and fourth ventricle (g,l) were examined by fl uorescent microscopy. The sections were stained with 300 n M DAPI for 10 min at room temperature. Green: GFP. Blue: cell nuclei. Original magnifi cation: × 100
A合EAR www.advmat.de demand for choline. High-affinity choline transporter-immu- Synthesis and Identify of DGL Derivatives and Derivative/DNA NPs noreactive cell bodies were demonstrated in these regions. 18) and Other Bioassay Parts: Experimental details for in vivo imaging of 10-modified NPs accumulated more in compound 10-BODIPY, synthesis and identify of DGL derivatives and nese regions and had higher GFP expression. To examine the derivative/DNA NPs, the cellular uptake mechanism of NP, transport v studies of NPs across the BCECs monolayer, in vivo imaging of NPs, transfection efficiencies quantitatively, the expression of pGl3 the distribution of gene expression in the mouse brain, and in vivo gene luciferase reporter vector)-control vector in brain was meas- quantitative expression are described in thethe Supporting Information ured( Figure 3b). The brain gene expression of DGL-PEG-10/ L3 was 8.63+0.61 x 10 light units per mg protein, which is 1.48-fold higher than that of DGL- PEG/pGL3 (5.85+0. 26x 10 light units per mg protein). The significant level (p) is smaller Supporting Information Supporting Information is available from the Wiley Online Library In summary, a series of novel bis-quaternary ammonium compounds with high BBB-ChT affinity have been synthesized Although BBB ChT has not been crystallized, the structure- activity relationships shown here could inspire design of BBB Acknowledgements ChT substrates with higher affinity. The sulfhydrylation form The authors thank Prof. Jianhua Zhu(School of Pharmacy, Fudan of the novel compound 7, i.e. compound 10, was utilized for University) for the 1251-labeled work. This work was supported by the the construction of the brain-targeting gene-delivery system. grant from National Basic Research And DGL-PEG-10/PDNA NPs demonstrated higher uptake effi- China(973program),National Natural Foundation of china iency in vitro and higher gene expression in vivo. The BBB (30973652), National Natural Science Four of China(81172993 ChT-mediated brain-targeting strategy was first employed in a and the "Key new drug creation program"2009ZX09310-006 drug-delivery system and proved to be an encouraging way to vercome the bbb Received: May 23, 2011 Published online: September 5, 2011 Experimental Section [ M. W. Smith, M. Gumbleton, /. Drug Targeting 2006, 14, 191 Synthesis of Compounds 5-8: Compounds a and 3b were prepared 2 R. Gabathuler, Neurobiol.Dis.2010,37,48 by dissolving 3, 5-dihydroxy-benzyl alcohol (1, 5 mmol) in acetonitril [3] W M. Pardridge, Drug Discovery Today 2007, 12, 54 adding 1, 3-dibromopropane(2a, 30 mmol) or 1, 4-dibromobutane [4]C. C. Visser, L. H. Voorwinden, D.J. Crommelin, M. Danhof, mmol), and K, CO,(30 mmol), then stirring the the solution under A.G. de boer. pharm. Res. 2004. 21.761 efilux for 4 h. The completion of the reactions was monitored by thin. [5] Z Laron, Arch. Physiol. Biochem. 2009, 175, yer chromatography (TLC). After filtration and removal of the solvent [65] Q.R. Smith, Adv. Exp. Med. Biol. 1993, 331, 83 via rotary evaporation, the crude products were purified by flash [7] M. Gynther, ). Ropponen, K. Laine, J. Leppanen, P. Haapakoski, L Peura, T. Jarvinen, ) Rautio, J. Med. Chem. 2009, 52, 3348 lentified by 'H NMR Compounds 5-8 were prepared by reacting an [8]W ). Geldenhuys, P.R. Lockman, T H Nguyen, C J. Van der Schyf, nylpyridine(4b) with compound 3a P. A. Crooks, L. P. Dwoskin, D. D. Allen, Bioorg. Med. Chem. 2005 3b for I h at 65C in the absence of solvent. The resulting solids were 13.4253. collected by filtration and purified by recrystallisation in ethanol [9] D. D. Allen, Q.R. Smith, /. Neurochem. 2001, 76, 1032. Cell Line and Animals: BCECs were kindly provided by Prof J. N Lou [o] S H Zeisel, J. Am. Coll. Nutr. 2004, 23, 621S (the Clinical Medicine Research Institute of the China-Japan Friendship [11D D. Allen, P. R Lockman, Life Sci.2003,73,1609 le, age 4-5 weeks, 20-25 g) were [12]). T. Ayers, L. P. Dwoskin, A. G. Deaciuc, V. P. Grinevich, ). Zhu, aintained under standard housing conditions. All animal experiments P. A. Crooks, Bioorg. Med. Chem. Lett. 2002, 12, 3067 e carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University [3]G. Zheng, Z Zhang, P. R Lockman, W.J. Geldenhuys, DD. Allen, Inhibition of PHl- Choline Chloride Uptake: Incubation buffer(0. 25 mL) L P. Dwoskin, P. A. Crooks, Bioorg. Med. Chem. Lett. 2010, 20, ontaining 10 nmol PHl- choline chloride dissolved in Hanks buffer wi different compounds at designated concentrations was added to ead [4] H. Co Martin, A. Papillaud, E. Souaid, H. Collet A. Commeyras, Biomacromolecules 2007, 8, 3235 served as a comparison. Incubation buffer without inhibitions was the [5 R.Q. Huang. S. Liu, K Shao, L Han, w. Ke, Y Liu, J.F. Li, SHuang incubation buffer and washing the cells three times with ice-cold Hanks [16] W L Ke, K. Shao, R. Q Huang, L Han, Y. Liu, ). F. Li, Y.Y Kuang fer. 2 N NaOH(0.2 L Y. Ye, N. Lou, C Jiang, Biomaterials 2009, 30, neutralized with 4 N HCI(O1 mL). Radioactivity was determined by [17 R Q- Huang, Y.H. Qu, W LKe, J.H. Zhu, Y.Y. Pei, C Jiang, FASEB ding scintillant liquid (2 mL) to the solubilized cell solution(0.1 mL) J2007,21,1117 and counting it in a liquid scintillation counter(LS 6000SE, Beckman, [18] H Misawa, K. Nakata, ). Matsuura, M. Nagao, T Okuda, T. Haga, euroscience 2001. 105. 87. 9 2011 WILEY-VCH Verlag GmbH Co KGaA, Weinheir Ad. Mater2011,23,4516-4520
4520 www.advmat.de www.MaterialsViews.com © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim COMMUNICATION wileyonlinelibrary.com Adv. Mater. 2011, 23, 4516–4520 demand for choline. High-affi nity choline transporter-immunoreactive cell bodies were demonstrated in these regions. [18] As a result, compound- 10- modifi ed NPs accumulated more in these regions and had higher GFP expression. To examine the transfection effi ciencies quantitatively, the expression of pGL3 (luciferase reporter vector)-control vector in brain was measured (Figure 3 b). The brain gene expression of DGL-PEG- 10 / pGL3 was 8.63 ± 0.61 × 10 3 light units per mg protein, which is 1.48-fold higher than that of DGL-PEG/pGL3 (5.85 ± 0.26 × 10 3 light units per mg protein). The signifi cant level ( p ) is smaller than 0.01. In summary, a series of novel bis-quaternary ammonium compounds with high BBB-ChT affi nity have been synthesized. Although BBB ChT has not been crystallized, the structure– activity relationships shown here could inspire design of BBB ChT substrates with higher affi nity. The sulfhydrylation form of the novel compound 7 , i.e., compound 10 , was utilized for the construction of the brain-targeting gene-delivery system. And DGL-PEG- 10 /pDNA NPs demonstrated higher uptake effi - ciency in vitro and higher gene expression in vivo. The BBB ChT-mediated brain-targeting strategy was fi rst employed in a drug-delivery system and proved to be an encouraging way to overcome the BBB. Experimental Section Synthesis of Compounds 5 – 8 : Compounds 3a and 3b were prepared by dissolving 3,5-dihydroxy-benzyl alcohol ( 1 , 5 mmol) in acetonitrile, adding 1,3-dibromopropane ( 2a , 30 mmol) or 1,4-dibromobutane ( 2b , 30 mmol), and K 2 CO 3 (30 mmol), then stirring the the solution under refl ux for 4 h. The completion of the reactions was monitored by thinlayer chromatography (TLC). After fi ltration and removal of the solvent via rotary evaporation, the crude products were purifi ed by fl ash chromatography on silica (1:4 ethyl acetate/petroleum ether eluent) and identifi ed by 1 H NMR. Compounds 5 – 8 were prepared by reacting an excess of isoquinoline ( 4a ) or 3-methylpyridine ( 4b ) with compound 3a or 3b for 1 h at 65 ° C in the absence of solvent. The resulting solids were collected by fi ltration and purifi ed by recrystallisation in ethanol. Cell Line and Animals : BCECs were kindly provided by Prof. J. N. Lou (the Clinical Medicine Research Institute of the China–Japan Friendship Hospital). Balb/c nude mice (male, age 4–5 weeks, 20–25 g) were maintained under standard housing conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. Inhibition of [ 3 H]-Choline Chloride Uptake : Incubation buffer (0.25 mL) containing 10 nmol [ 3 H]-choline chloride dissolved in Hanks buffer with different compounds at designated concentrations was added to each well and incubated at 37 ° C for 10 min. The unlabeled choline chloride served as a comparison. Incubation buffer without inhibitions was the total uptake as a control. The uptake was stopped by aspirating the incubation buffer and washing the cells three times with ice-cold Hanks buffer. 2 N NaOH (0.2 mL) was added in each well to lyse the cells and neutralized with 4 N HCl (0.1 mL). Radioactivity was determined by adding scintillant liquid (2 mL) to the solubilized cell solution (0.1 mL) and counting it in a liquid scintillation counter (LS 6000SE, Beckman, USA). Synthesis and Identify of DGL Derivatives and Derivative/DNA NPs and Other Bioassay Parts : Experimental details for in vivo imaging of compound 10-BODIPY, synthesis and identify of DGL derivatives and derivative/DNA NPs, the cellular uptake mechanism of NPs, transport studies of NPs across the BCECs monolayer, in vivo imaging of NPs, the distribution of gene expression in the mouse brain, and in vivo gene quantitative expression are described in the the Supporting Information. Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements The authors thank Prof. Jianhua Zhu (School of Pharmacy, Fudan University) for the 125 I-labeled work. This work was supported by the grant from National Basic Research Program (2007CB935802) of China (973program), National Natural Science Foundation of China (30973652), National Natural Science Foundation of China (81172993), and the “Key new drug creation program” 2009ZX09310-006. Received: May 23, 2011 Published online: September 5, 2011 [ 1 ] M. W. Smith , M. Gumbleton , J. Drug Targeting 2006 , 14 , 191 . [ 2 ] R. Gabathuler , Neurobiol. Dis. 2010 , 37 , 48 . [ 3 ] W. M. Pardridge , Drug Discovery Today 2007 , 12 , 54 . [ 4 ] C. C. Visser , L. H. Voorwinden , D. J. Crommelin , M. Danhof , A. G. de Boer , Pharm. Res. 2004 , 21 , 761 . [ 5 ] Z. Laron , Arch. Physiol. Biochem. 2009 , 115 , 112 . [ 6 ] Q. R. Smith , Adv. Exp. Med. Biol. 1993 , 331 , 83 . [ 7 ] M. Gynther , J. Ropponen , K. Laine , J. Leppänen , P. Haapakoski , L. Peura , T. Järvinen , J. Rautio , J. Med. Chem. 2009 , 52 , 3348 . [ 8 ] W. J. Geldenhuys , P.R. Lockman , T. H. Nguyen , C. J. Van der Schyf , P. A. Crooks , L. P. Dwoskin , D. D. Allen , Bioorg. Med. Chem. 2005 , 13 , 4253 . [ 9 ] D. D. Allen , Q. R. Smith , J. Neurochem. 2001 , 76 , 1032 . [ 10 ] S. H. Zeisel , J. Am. Coll. Nutr. 2004 , 23 , 621S . [ 11 ] D. D. Allen , P. R. Lockman , Life Sci. 2003 , 73 , 1609 . [ 12 ] J. T. Ayers , L. P. Dwoskin , A. G. Deaciuc , V. P. Grinevich , J. Zhu , P. A. Crooks , Bioorg. Med. Chem. Lett. 2002 , 12 , 3067 . [ 13 ] G. Zheng , Z. Zhang , P. R. Lockman , W. J. Geldenhuys , D. D. Allen , L. P. Dwoskin , P. A. Crooks , Bioorg. Med. Chem. Lett. 2010 , 20 , 3208 . [ 14 ] H. Cottet , M. Martin , A. Papillaud , E. Souaïd , H. Collet , A. Commeyras , Biomacromolecules 2007 , 8 , 3235 . [ 15 ] R. Q. Huang , S. Liu , K. Shao , L. Han , W. Ke , Y. Liu , J. F. Li , S. Huang , C. Jiang , Nanotechnology 2010 , 21 , 265101 . [ 16 ] W. L. Ke , K. Shao , R. Q. Huang , L. Han , Y. Liu , J. F. Li , Y. Y. Kuang , L. Y. Ye , J. N. Lou , C. Jiang , Biomaterials 2009 , 30 , 6976 . [ 17 ] R. Q. Huang , Y. H. Qu , W. L. Ke , J. H. Zhu , Y. Y. Pei , C. Jiang , FASEB J. 2007 , 21 , 1117 . [ 18 ] H. Misawa , K. Nakata , J. Matsuura , M. Nagao , T. Okuda , T. Haga , Neuroscience 2001 , 105 , 87