A leptin derived 30-amino-acid peptide modified pegylated ... · A leptin derived 30-amino-acid...

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A leptin derived 30-amino-acid peptide modied pegylated poly-L-lysine dendrigraft for brain targeted gene delivery Yang Liu a , Jianfeng Li a , Kun Shao a , Rongqin Huang a , Liya Ye b , Jinning Lou b , Chen Jiang a, * a Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China b Institute of Clinical Medical Sciences, ChinaeJapan Friendship Hospital, The ministry of Health, 2 East Yinghua Road, Beijing 100029, PR China article info Article history: Received 11 January 2010 Accepted 4 March 2010 Available online 10 April 2010 Keywords: Brain targeting Gene delivery Leptin Non-viral vector Dendrigraft poly-L-lysine abstract The bloodebrain barrier is the major obstacle that prevents diagnostic and therapeutic drugs being delivered to the central nervous systems in order to exert their effects. Specic ligand-receptor binding mediated endocytosis is one of the possible strategies to cross this barrier. A 30-amino-acid peptide (leptin30) derived from an endogenic hormonedleptin is exploited as brain-targeting ligand as it is reported to possess the same brain accumulation efciency after intravenous injection. Dendrigraft poly- L-lysine (DGL) is used as non-viral gene vector in this study. DGLePEGeLeptin30 was complexed with plasmid DNA yielding nanoparticles (NPs). The cellular uptake characteristic and mechanism were explored in brain capillary endothelial cells (BCECs) which express leptin receptors. Furthermore, brain parenchyma microglia cells such as BV-2 cells expressing leptin receptors could promote ligand-receptor mediated endocytosis leading to enhanced gene transfection ability of DGLePEGeLeptin30/DNA NPs. The targeted NPs were proved to be transported across in vitro BBB model effectively and accumulate more in brains after i.v. resulting in a relatively high gene transfection efciency both in vitro and in vivo. Besides, the NPs showed low cytotoxicity after in vitro transfection. Thus, DGLePEGeLeptin30 provides a safe and noninvasive approach for the delivery of gene across the bloodebrain barrier. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Central nervous system diseases such as Alzheimers disease (AD), Parkinsons disease (PD) and brain tumors have been major threats to human health [1e3]. Though great efforts have been made during the last thirty years, there is still much to be done to develop efcient and safe therapies [4,5]. Comparing with the traditional chemical drugs, the gene therapy holds great promise in curing brain diseases in both laboratory and clinical applications [6,7]. One of the principal concerns about the gene therapy is the gene vectors. The ideal gene vector should possess such characteristics as high gene loading dose, low immunogenicity, better shelf-life, the possibility of repeated administration and being apt to further modication. Despite viral gene vectors have been proved to be efcient enough, the safety consideration has somehow prevented their further applications [8]. Among the non-viral approaches, synthetic polymers such as PEI, PLGA, PAMAM have been inten- sively investigated in gene delivery system [9e11]. PAMAM have been successfully applied to non-viral gene delivery systems in our previous studies [12e14]. The critical concerns about the synthetic polymers include safety and efciency. Dendrigraft poly-L-lysine (DGL) have emerged as a new kind of synthetic polymers consisted of lysine [15e17] and herein been employed as gene vector due to its degradability and rich external amino groups. The dendrimers used in this study were DGL Generation 3 with 123 amino groups per molecular. Besides their biodegradability [17], the external amino groups could encapsulate DNA through electric interactions. Furthermore, they could be modied with PEG and targeting ligand rendering vectors long circulation and targeting properties. Another major obstruction of brain gene therapy is the restrict conjunction of the bloodebrain barrier (BBB). Several strategies have been developed to overcome the BBB [12e14,18e20]. Among these, cell-penetrating peptides (CPPs) are a group of short peptides with the potent ability to translocate across the BBB [18] and have been studied intensively during the last decades as materials well- suitable for the development of drug delivery vehicles [21]. However, because of lack of selectivity, the cell-penetrating peptide modied drug delivery system would cause unnecessary uptake by disease unrelated cells or tissues. Receptor-mediated endocytosis is one of the major mechanisms by which various agents can cross the BBB. The drug or gene delivery systems modied with ligands such as transferrin and lactoferricin, could be uptaken by brain capillary * Corresponding author. Tel./fax: þ86 21 5198 0079. E-mail address: [email protected] (C. Jiang). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.03.011 Biomaterials 31 (2010) 5246e5257

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Biomaterials 31 (2010) 5246e5257

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

A leptin derived 30-amino-acid peptide modified pegylated poly-L-lysinedendrigraft for brain targeted gene delivery

Yang Liu a, Jianfeng Li a, Kun Shao a, Rongqin Huang a, Liya Ye b, Jinning Lou b, Chen Jiang a,*

aDepartment of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR Chinab Institute of Clinical Medical Sciences, ChinaeJapan Friendship Hospital, The ministry of Health, 2 East Yinghua Road, Beijing 100029, PR China

a r t i c l e i n f o

Article history:Received 11 January 2010Accepted 4 March 2010Available online 10 April 2010

Keywords:Brain targetingGene deliveryLeptinNon-viral vectorDendrigraft poly-L-lysine

* Corresponding author. Tel./fax: þ86 21 5198 0079E-mail address: [email protected] (C. Jiang)

0142-9612/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.biomaterials.2010.03.011

a b s t r a c t

The bloodebrain barrier is the major obstacle that prevents diagnostic and therapeutic drugs beingdelivered to the central nervous systems in order to exert their effects. Specific ligand-receptor bindingmediated endocytosis is one of the possible strategies to cross this barrier. A 30-amino-acid peptide(leptin30) derived from an endogenic hormonedleptin is exploited as brain-targeting ligand as it isreported to possess the same brain accumulation efficiency after intravenous injection. Dendrigraft poly-L-lysine (DGL) is used as non-viral gene vector in this study. DGLePEGeLeptin30 was complexed withplasmid DNA yielding nanoparticles (NPs). The cellular uptake characteristic and mechanism wereexplored in brain capillary endothelial cells (BCECs) which express leptin receptors. Furthermore, brainparenchyma microglia cells such as BV-2 cells expressing leptin receptors could promote ligand-receptormediated endocytosis leading to enhanced gene transfection ability of DGLePEGeLeptin30/DNA NPs.The targeted NPs were proved to be transported across in vitro BBB model effectively and accumulatemore in brains after i.v. resulting in a relatively high gene transfection efficiency both in vitro and in vivo.Besides, the NPs showed low cytotoxicity after in vitro transfection. Thus, DGLePEGeLeptin30 providesa safe and noninvasive approach for the delivery of gene across the bloodebrain barrier.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Central nervous system diseases such as Alzheimer’s disease(AD), Parkinson’s disease (PD) and brain tumors have been majorthreats to human health [1e3]. Though great efforts have beenmadeduring the last thirty years, there is still much to be done to developefficient and safe therapies [4,5]. Comparing with the traditionalchemical drugs, the gene therapy holds great promise in curing braindiseases in both laboratory and clinical applications [6,7].

One of the principal concerns about the gene therapy is the genevectors. The ideal gene vector should possess such characteristics ashigh gene loading dose, low immunogenicity, better shelf-life, thepossibility of repeated administration and being apt to furthermodification. Despite viral gene vectors have been proved to beefficient enough, the safety consideration has somehow preventedtheir further applications [8]. Among the non-viral approaches,synthetic polymers such as PEI, PLGA, PAMAM have been inten-sively investigated in gene delivery system [9e11]. PAMAM havebeen successfully applied to non-viral gene delivery systems in our

..

All rights reserved.

previous studies [12e14]. The critical concerns about the syntheticpolymers include safety and efficiency. Dendrigraft poly-L-lysine(DGL) have emerged as a new kind of synthetic polymers consistedof lysine [15e17] and herein been employed as gene vector due toits degradability and rich external amino groups. The dendrimersused in this study were DGL Generation 3 with 123 amino groupsper molecular. Besides their biodegradability [17], the externalamino groups could encapsulate DNA through electric interactions.Furthermore, they could be modified with PEG and targeting ligandrendering vectors long circulation and targeting properties.

Another major obstruction of brain gene therapy is the restrictconjunction of the bloodebrain barrier (BBB). Several strategieshave been developed to overcome the BBB [12e14,18e20]. Amongthese, cell-penetrating peptides (CPPs) are a group of short peptideswith the potent ability to translocate across the BBB [18] and havebeen studied intensively during the last decades as materials well-suitable for the development of drug delivery vehicles [21].However, because of lack of selectivity, the cell-penetrating peptidemodified drug delivery systemwould cause unnecessary uptake bydisease unrelated cells or tissues. Receptor-mediated endocytosis isone of themajor mechanisms bywhich various agents can cross theBBB. The drug or gene delivery systems modified with ligands suchas transferrin and lactoferricin, could be uptaken by brain capillary

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endothelial cells (BCECs) and cross BBB via endocytosis pathwaymediated by the specific ligand-receptor binding [12,22]. Comparedwith the above largemolecule protein ligands, short peptide ligandshave the advantages of a relatively small molecular weights, beingeasily synthesized, relatively low cytotoxicity and immunogenicity,and degradability [18]. There have been quite a few research papersabout the short peptide ligands modified drug delivery systemsrecently. Andwe have previously reported a 29 amino acids peptidederived from rabies virus glycoprotein could serve as brain-target-ing ligand inducing efficient brain-targeting gene delivery [13]. Onthe basis of the above evidence, a 30 amino acids peptide derivedfrom leptin was investigated in this research.

Leptin, a 146 amino acid polypeptide secreted into the blood-stream by adipocytes, acts centrally on leptin receptor-expressingcells located in the hypothalamus and other parts of the brain,leading to decreased food intake and retardingweight gain [23,24]. Ithas been widely reported that leptin could be taken up by the brain[25,26]. Considering the 16 kDa molecular weight of leptin precludethe possibility of passive diffusion across the bloodebrain barrier,a process that provides limited entry for some relatively smallmolecules, the leptin receptor mediated transport is believed to beone of the possiblemechanisms besides leptin receptor independentpathway. Barrett’s group has identified several leptin derivedpeptides that are taken up by the brain [27]. Among these, peptidecorresponded to position 61e90 (leptin30, 30 amino acids) wasobserved to possess the highest brain: plasma ratio which wasequivalent to that of leptin. For leptin30, the majority of the radio-activity was localized more to parenchyma than capillaries. As theregions conferring brain uptake correspond closely to the sequencesthat are necessary for receptor binding, the results reinforce the viewthat the receptor pathway plays a large part in leptin uptake [27].

Since leptin is known to be taken up into all regions of the brain[28,29], leptin30 could be exploited as the brain-targeting ligandmodified on the surface of gene vectors. Furthermore, it could bededuced that after transcytosis across the BBB, leptin30 couldenhance internalization to the brain parenchyma cells by thespecific ligand-receptor mediated endocytosic pathway. Therefore,leptin30 modified gene delivery system may have significantrepercussions for brain diseases therapeutics.

In this study, leptin30 was modified by covalent linkage bond onthe surface of DGL through bifunctional polyethyleneglycol (PEG),yielding DGLePEGeleptin30. The brain-targeting efficiency ofDGLePEGeleptin30 as gene delivery vectors was evaluated in vitroand in vivo. Furthermore, the uptake mechanism ofDGLePEGeLeptin30/DNA NPs was explored.

2. Materials and methods

2.1. Materials

DGL were purchased from Colcom, France. a-Malemidyl-u-N-hydrox-ysuccinimidyl polyethyleneglycol (NHS-PEG-MAL, MW 3400) was obtained fromNektar Therapeutics (Huntsville, AL, USA). The peptide leptin30with a cysteine on C-terminal (YQQVLTSLPSQNVLQIANDLENLRDLLHLLC) was synthesized by GL biochem(Shanghai) Ltd. The red fluorescent protein (RFP) plasmid (Shanghai GeneChem Co.,Ltd, China) and pGL2-control vector (Promega, Madison, WI, USA) were purifiedusing QIAGEN PlasmidMega Kit (Qiagen GmbH, Hilden, Germany). Filipin, colchicineand were purchased from SigmaeAldrich (St. Louis, MO, USA). EMA and 4,6-dia-midino-2-phenylindole (DAPI) were purchased from Molecular Probes (Eugene, OR,USA). Lipofectamine2000 was bought from Invitrogen. Male ICR mice (4e5 wk old)of 20e25 g body weight and nude mice of 20e25 g body weight were purchasedfrom the Sino-British SiPPR/BK Lab. Animal Ltd and maintained under standardhousing conditions. All animal experiments were carried out in accordance withguidelines evaluated and approved by the ethics committee of Fudan University.

2.2. Cell culture

The primary cultured brain capillary endothelial cells (BCECs) isolated fromBALB/C mouse were kindly provided by Prof. J. N. Lou (the Clinical Medicine

Research Institute of the ChineseeJapanese Friendship Hospital). Primary BCECswere cultured as described previously [30]. Briefly, BCECs were expanded andmaintained in special Dulbecco’s modified Eagle medium (DMEM) (SigmaeAldrich)supplementedwith 20% heat-inactivated fetal calf serum (FCS),100 mg/ml epidermalcell growth factor, 2 mmol/L L-glutamine, 20 mg/ml heparin, 40 mU/ml insulin, 100 U/ml penicillin, and 100 mg/ml streptomycin and cultured at 37 �C under a humidifiedatmosphere containing 5% CO2. All cells used in this study were between passage 15and passage 30.

BV-2 microglia cells were gifts from Prof. L.Y. Feng (Shanghai Institute ofmaterial medica, Chinese academy of sciences). BV-2 cells were cultured in DMEM(high sucrose), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillinand 100 mg/ml streptomycin at 37 �C in a humidified 5% CO2 incubator. All cells usedin this study were between passage 9 and passage 20.

2.3. Synthesis of DGL derivatives

DGL was reacted with NHS-PEG3400-MAL at different molar ratio in PBS (pH8.0) for 2 h at room temperature. The primary amino groups on the surface of DGLwere specifically reacted with the NHS groups of the bifunctional PEG derivative.The resulting conjugate, DGLePEG, was purified by ultrafiltration througha membrane (cutoff� 5 kDa) and the buffer was changed to PBS (pH 7.0). ThenDGLePEG was reacted with peptide leptin30 in PBS (pH 7.0) for 24 h at roomtemperature. The MAL groups of DGLePEG were specifically reacted with the thiolgroups of Leptin30. Ethidium monoazide (EMA) is a fluorescent photoaffinity labelthat, after photolysis, binds covalently to plasmid DNA. Thus, after complexing withthe EMA-labeled plasmid DNA, the DGL series NPs was fluorescent. For synthesis ofEMA-labeled DNA, EMA was labeled on DNA through UV exposure 1 h and seriespurification.

2.4. Characterization of DGLePEGeLeptin30

The characteristics of DGLePEGeLeptin30 were analyzed by nuclear magneticresonance (NMR) spectroscopy. Basically, DGLePEGeLeptin30 was freeze-dried,solubilized inD2Oandanalyzed ina400 MHzspectrometer (Varian,PaloAlto,CA,USA).

2.5. Preparation and characterization of dendrimers/DNA nanoparticles(NPs)

Dendrimers (DGL, DGLePEG, and DGLePEGeLeptin30) were freshly preparedand diluted to appropriate concentrations in PBS (pH 7.4). DNA solution (100 mgDNA/ml 50 mM sodium sulfate solution) was added to obtain specified weightratios (6:1, DGL to DNA, w/w) and immediately vortexed for 30 s at roomtemperature. Freshly prepared NPs were used in the experiments that follow. Themean diameter and zeta-potential of DGL series vectors (DGL, DGLePEG,DGLePEGeLeptin30)/DNA NPs were determined by dynamic light scatteringusing a Zeta-Potential/Particle Sizer Nicomp� 380 ZLS (PSS Nicomp Particle SizeSystem, U.S.A.).

2.6. Electrophoresis of dendrimers/DNA NPs and their DNA protection assay

The three NPs including DGL/DNA, DGLePEG/DNA and DGLePEGeLeptin30/DNAwere freshly prepared with DGL to DNA at weight ratio of 6:1. 0.7% agarose gelelectrophoresis was performed to evaluate the DNA encapsulation effect caused byDGL in the NPs comparedwith naked DNA. To determine the DNase I stability, DNaseI (Takara Bio, Japan) (1 U/mg of DNA) was added to each NPs, and the mixtures wereincubated at 37 �C for 30 min. 250 mM EDTA solution was added and incubated atroom temperature for 10 min to inactivate the DNase I. After that, 15 mg/ml sodiumheparin was added and incubated at room temperature for 2 h to release the DNAfrom the NPs. All the samples were analyzed by 0.7% agarose gel electrophoresis. Theintegrity of the plasmid in each sample was compared with untreated naked DNAand DNase I treated naked DNA.

2.7. Cellular uptake of dendrimers/DNA NPs and with different endocytosisinhibitors

BCECs were seeded at a density of 2�104 cells/well in 24-well plates (Corning-Coaster, Tokyo, Japan), incubated for 72 h, and checked under the microscope forsimilar confluency and morphology. After this, BCECs were incubated with EMA-labeled DGLePEG/DNA or DGLePEGeLeptin30/DNA NPs at the concentration of20 mg/well measured by DGL in the present of serum-freemedium for 1 h at 37 �C. Incase of inhibitors groups, different inhibitors were pre-incubated with cells 10 min,following the addition of the DGLePEGeLeptin30/DNA NPs treated at the conditionmentioned above.

2.8. Scatchard analysis of leptin receptor-binding assay

BCECs were seeded at a density of 2�104 cells/well in 24-well plates, incubatedfor 72 h, and checked under the microscope for similar confluency and morphology.DGL was iodinated as described previously with radioactivity of 1 mCi/mg 125I-DGL[31]. Radiolabeled DGL was applied to synthesize DGLePEGeLeptin30. BCECs were

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incubated with 125I-DGLePEGeLeptin30 at concentrations from 0.2 to 2500 nM

(leptin30) at 4 �C for 2 h. After that, the contents of each incubation well wereremoved and washed with cold PBS 7.4 for three times. 200 ml 1 M NaOHwere addedto lysis cells for 3 h. 100 ml 2 M HCl was used to neutralize NaOH. The radioactivity ofthe final well contents was analyzed by a g-counter (SN-695, China). The aboveexperiments were performed to determine the total binding of vector with leptinreceptors. Nonspecific binding was determined by adding 5 mM nonradioactiveDGLePEGeLeptin30 to each reaction mixture in a parallel assay. The amount ofcellular protein was determined by a BCA Protein Assay. For each concentration,experiments were repeated for 4 times.

2.9. Transport studies of NPs across BCECs monolayer

BCECs were seeded at a density of 6�103 cells/cm2 onto polycarbonate 24-wellTranswell filters of 1.0 mm mean pore size, 0.33 cm2 surface area (FALCON CellCulture Insert, Becton Dickinson Labware, U.S.A.). After about 3 days’ culture, thecells were checked under the microscope for complete confluency. The cell mono-layer integrity was monitored using an epithelial voltohmmeter (MILLICELL�-ERS,Millipore, U.S.A.) to measure the transendothelial electrical resistance (TEER). Onlycell monolayers with TEER exceeding 200 U cm2 were selected for this experiment[32].

Radiolabeled DGL was applied to synthesize DGLePEG/DNA andDGLePEGeLeptin30/DNA NPs. 300 ml fetal bovine serum (FBS)-free medium con-taining 0.6 mCi 14C-sucrosewas added to the donor chamber at time 0, tomonitor theintegrity of BCECs monolayers. At the same time, BCECs monolayers were treatedwith DGL/DNA and DGLePEGeLeptin30/DNA NPs, with a final amount of 20 mg DGL/well, separately. The incubation was performed at 37 �C on a rocking platform at50 rpm. The radioactivity of 125I in each aliquot was assessed using a g-counter(SN-695, China). After that, 10 ml from the 20 ml aliquot was dissolved in 1 ml ofscintillation cocktail, and analyzed in a liquid scintillation counter (Packard Tri-Carb,GMI, U.S.A.). The correction factor of 14C-sucrose was 25% for the equipment. Theapparent permeability (Papp) was calculated according to Irvine et al. [33] as follows:

Papp ¼ ðdQ=dtÞ � ð1=C0Þ � ð1=AÞwhere dQ/dt is the permeability rate (nmol/s), C0 is the initial concentration (nmol/ml) in the donor chamber, and A is the surface area (cm2) of the membrane filter.

Both TEER and permeability of 14C-sucrose of BCECs monolayers were measuredfrom time 0e60 min to monitor the integrity of monolayers [35]. Basically, thecleared volume of 14C-sucrose was plotted vs. time, and the slope was estimated bylinear regression analysis to give themean and standard error of the estimate. The PS(permeability$surface area product) value for the BCECs monolayer, called PSec (cm/min), was computed as follows:

1=PSec ¼ 1=PSt � 1=PSf

where PSt is the PS value for the BCECsmonolayer and the filter, PSf is the PS value forthe filter only.

2.10. In vitro transfection experiment

BV-2 cells were seeded at a density of 5�104 cells/well in a 24-well plate ina DMEM medium containing 10% FBS, and grown to reach 70e80% confluence priorto transfection. Before transfection, the medium was exchanged with fresh serum-free medium. The cells were treated with different NPs solutions containing 5 mg ofplasmid DNA for 4 h at 37 �C. After exchange with a fresh serum-containingmedium, cells were further incubated for 2 days after transfection. 5 mg plasmid DNAmixed with Lipofectamine2000 according to the standard protocol as described ininstruction served as positive control. In the case of qualitative evaluation, the redfluorescence images were taken using a fluorescence microscope. For luciferaseactivity assay, medium was removed and the cells were rinsed with DPBS andshaken for 30 min at room temperature in 150 ml of luciferase cell culture lysisreagent supplied by the Promega Luciferase Assay Kit. The lysis solution wascentrifuged at 14,000g for 2 min at 4 �C. Luciferase activity was measured by BPCL-1Ultra-Weak CL and BL Analyzer and total amount of cellular proteinwas determinedby a BCA Protein Assay. The final results were reported in terms of relative light units(RLU)/mg protein.

2.11. Cytotoxicity assay

The cytotoxicity of the polymers was measured by MTT assay. BCECs wereseeded in a 96-well tissue culture plate at 5000 cells per well in 90 ml DMEMmedium containing 10% FBS. Cells achieving 70e80% confluence after 24 h wereexposed to 100 ml of different NPs solutions with various concentrations and Lip-ofectamine200 at equivalent to 0.2 mg DNA per well for 2 h. Then, 100 ml of stocksolution of MTT (1 mg/ml in Hank’s) was added to each well. After 3 h of incubationat 37 �C, each medium was removed and 100 ml of DMSO was added to each well todissolve the formazan crystals formed by proliferating cells. Cells without treatmentwere used as a control. Absorbance was measured at 570 nm using a microplate

reader (BIO-TEK, MQX200R PowerWave� XS). Cell viability of each group wasexpressed as a percentage relative to that of control cells.

The same experiments were also performed on BV-2 cells. For 48 h assay, DMEMwas added after 2 h incubation with NPs.

2.12. In vivo imaging analysis

The DGLePEGeLeptin30/DNA NPs labeled by EMA were injected into the tailvein of nude mice at dose of 50 mg DNA/mouse. Then, the mice were anesthetized.Images were taken by CRI in vivo imaging system 60 min after injection. Brains wereextirpated 2 h after injection for comparing the relative accumulation. EMA-labeledDGL/DNA NPs served as negative control.

2.13. Distribution of gene expression qualitatively in mouse brain

TheDGLePEGeLeptin30/pRFP NPs (6:1, DGL to DNA, w/w) as well as DGL/pRFPand DGLePEG/pRFP NPs were injected into the tail vein of mice at a dose of 50 mgDNA/mouse. About 48 h later, animals were anesthetized with diethyl ether andkilled by decapitation. The brains were removed, fixed in 4% paraformaldehyde for48 h, placed in 15% sucrose solution until subsidence (6 h), then in 30% sucroseuntil subsidence (24 h). After this, brains were frozen in OCT embedding medium(Sakura, Torrance, CA, USA) at 80 �C. Frozen sections of 20 mm thickness wereprepared with a cryotome Cryostat (Leica, CM 1900, Wetzlar, Germany) andstained with 300 nM DAPI for 10 min at room temperature. After washing twicewith PBS (pH 7.4), the sections were immediately examined under the fluorescencemicroscope.

2.14. In vivo gene quantitative expression

The DGLePEGeLeptin30/pGL2 NPs (6:1, DGL to DNA, w/w) were injected intothe tail vein of mice at a dose of 50 mg DNA/mouse. At 48 h after injection, the micewere humanely decapitated and the principal organs (including brain, heart, liver,spleen, lung, and kidney) were extirpated. The organs were carefully washed withdistilled water and homogenized in 1 ml lysis reagent (Promega, Madison, WI, USA)using a JY92-IIN tissue homogenizer. The homogenate was centrifuged at 14,000gfor 20 min at 4 �C. Luciferase activity and cellular proteins in the supernatant werequantified by a Luciferase Assay System and total amount of cellular protein wasdetermined by a BCA Protein Assay, respectively. The results were expressed as lightunits/mg protein. Meanwhile DGL/pGL2 and DGLePEG/pGL2 NPs were also injectedfor comparison.

3. Results

3.1. Characterization of DGLePEGeLeptin30 andDGLePEGeLeptin30/DNA NPs

In NMR spectra, the solvent peak of D2O was found at 4.65 ppm.The methylene protons of branching units of DGL and a series ofprotons of leptin30 peptide have multiple peaks between 4.3 and1.1 ppm. The NMR spectrum of DGLePEG had a characteristic peakof theMAL group in PEG at 6.7 ppm (data not shown). TheMAL peakdisappeared in the NMR spectrum of DGLePEGeleptin30, whereasthe repeat units of PEG still presented a sharp peak at 3.6 ppm(Fig.1) showing that theMAL grouphad reactedwith the thiol groupof peptide leptin30. The NMR spectra result proved the existence ofthe conjugate structure of DGLePEGeleptin30. Meanwhile Ellman’sreagent was used to determine the percentage of unreacted thiol.And little thiol was detected (data not shown) which could beexplained as the thiol at the end of leptin30 reactedwith theMAL atone end of PEG specifically. The particle size and zeta-potentialweremeasured. The results showed the vector/pDNA complex was nano-scaled (118� 69 nm, 114� 49 nm, 141�33 nm for DGL/DNA,DGLePEG/DNA and DGLePEGeLeptin30/DNA NPs, respectively)and positively charged (þ1.30� 0.56 mV, þ0.87� 0.43 mV,þ1.15� 0.47 mV for DGL/DNA, DGLePEG/DNA andDGLePEGeLeptin30/DNA NPs, respectively).

3.2. Electrophoresis results

Fig. 2A showed the electrophoresis results of DGL series vector/DNA NPs. All three NPs (DGL/DNA, DGLePEG/DNA andDGLePEGeLeptin30/DNA) with DGL to DNA at weight ratio of 6:1

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Fig. 1. NMR spectra of DGL-PEG-leptin30 in D2O at 400 MHz.

Y. Liu et al. / Biomaterials 31 (2010) 5246e5257 5249

could encapsulate DNA completely with no electrophoresis shift(Fig. 2A lane 3e5) compared with naked DNA (Fig. 2A lane 2). Toinvestigate whether DGL series vectors could protect DNA againstenzymatic degradation, naked DNA and the three DGL series NPswere incubated with DNase I. For all the three NPs, no electropho-resis shifts were observed after that (data not shown). To check theintegrity of plasmid DNA in NPs after DNase I incubation, sodiumheparinwas added to releaseDNA. Fig. 2B shows that naked plasmidDNA (lane 2) was completely digested by DNase I, whereas all thethree NPs (lane 3e5) could protect DNA from digestion.

3.3. Uptake comparison of DGL series vector/DNA NPs by BCECs invitro

The EMA-labeled DGL series vector/DNA NPs were used toinvestigate cellular uptake characteristics. The results were shownqualitatively using fluorescent images. DGL with different PEGyla-tion and leptin30 brain-targeting modification were synthesized asthe gene vectors. The uptake efficiency was investigated in BCECs todetermine an appropriate proportion for the subsequent experi-ments. The fluorescence intensity exhibited by BCECs had slightly

Fig. 2. A: Agarose gel electrophoresis was performed. Lane 1: marker; lane 2: naked DNA; laThe stability of DNA-loaded NPs against DNase I digestion. Plasmid DNAwas released from thDNase I incubation. Lane 1: naked plasmid DNA; lane 2: naked plasmid DNA treated withtreatment of heparin after DNase I incubation.

increased when cells were treated with the EMA-labeledDGLePEGeleptin30/DNA NPs at 1:5:2 (DGL:PEG:Leptin30, weightratio) and 1:10:3 (Fig. 3C and D) compared with DGL/DNA NPs andDGLePEGeleptin30/DNA (1:2:1) (Fig. 3A and B). As the ratio 1:5:2and 1:10:3 showed no significant difference in uptake, DGL:PEG:Leptin30 at the weight ratio of 1:5:2 was selected and studied infurther experiments from the economic consideration.

3.4. The exploration of NPs’ cell uptake mechanism

As shown in Fig. 4AeC, the BCECs uptake of DGLePEGeleptin30/DNA NPs was higher than that of DGL/DNA and DGLePEG/DNA NPs.The fluorescence intensity of all the three DGL series vector/DNANPs declined dramatically at 4 �C (Fig. 4DeF) compared withtreated at 37 �C (Fig. 4AeC). Excessive free leptin30 could inhibitthe uptake of DGLePEGeleptin30/DNA NPs (Fig. 4G), but had littleinfluence on that of non-leptin30 modified NPs (Fig. 4H and I).Different endocytosic pathway inhibitors were used to clarify theendocytosis process. Treatment with transferrin resulted ina decrease of fluorescent intensity (Fig. 4K and L) of the DGLePEG/DNA and DGLePEGeLeptin30/DNA NPs uptake while showed no

ne 3: DGL/DNA NPs; lane 4: DGL-PEG/DNA NPs; lane 5: DGL-PEG-Leptin30/DNA NPs. B:e NPs by the addition of sodium heparin separated by agarose gel electrophoresis afterDNase I; lane 3e5: DGL/DNA, DGL-PEG/DNA, and DGL-PEG-leptin30/DNA NPs with

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Fig. 3. Cellular uptake of the DGL-PEG-Leptin30/DNA NPs at different synthesis ratio was examined by fluorescent microscopy after a 60-min incubation. A: DGL/DNA NPs. B: DGL-PEG-Leptin30 (weight ratio¼ 1:2:1)/DNA NPs. C: DGL-PEG-Leptin30 (weight ratio¼ 1:5:2)/DNA NPs. D: DGL-PEG-Leptin30 (weight ratio¼ 1:10:3)/DNA NPs. Red: EMA-labeledDNA. Original magnification: �200.

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effect on the uptake of DGL/DNA NPs (Fig. 4J). And the fluorescentintensity of DGL/DNA and DGLePEGeLeptin30/DNA NPs declinedto some extent (Fig. 4M and O) by filipin. The treatment of colchi-cine seemed to have little influence on the DGLePEG/DNA NPsuptake (Fig. 4Q). However, the fluorescent intensity of DGL/DNAand DGLePEGeLeptin30/DNA NPs decreased with the pre-incu-bation of colchicine (Fig. 4P and R).

3.5. Scatchard analysis of leptin receptor-binding assay

Binding of 125I-DGLePEGeLeptin30 to BCECs was saturable inthe presence of excess of nonradiolabeled DGLePEGeLeptin30 inboth low (Fig. 5, main graph A) and high (Fig. 5, main graph B)concentration ranges. Scatchard analysis of the binding data (Fig. 5,inset) indicated the presence of two independent leptin bindingsites. The parameters governing the binding of DGLePEGeLeptin30to the BCECs, estimated by nonlinear regression analysis, indicateda high-affinity binding site (Fig. 5, inset A) with a dissociationconstant Kd

1 of 1.737� 0.21 nM (estimate� SD) compared with thatof leptin (5.1�2.8 nM reported by Pardridge [25]) and bindingmaximum (Bmax

1 ) of 0.6258� 0.02 pmol/mg pro. (compared withleptin, 0.34� 0.16 pmol/mg pro. reported). While, low-affinity andhigh-capacity class of binding sites (Fig. 5, inset B) had an estimateddissociation constant (Kd

2) of 421.8� 66.13 nM (leptin, 743� 226 nM

reported) and binding maximum (Bmax2 ) of 372.1�18.24 pmol/mg

pro. (leptin, 26� 5 pmol/mg pro. reported).

3.6. Transport studies of NPs across BCECs monolayer

The results of transport studies of DGL/DNA andDGLePEGeLeptin30/DNA NPs were shown in Fig. 6A. Papp ofDGLePEGeLeptin30/DNA NPs was significantly higher than that ofDGL/DNA and DGLePEG/DNA NPs after 10 min. The transendothelialelectrical resistance (TEER) showed no significant difference fromthat of controls (data not shown). The permeability of 14C-sucrose of

BCECs monolayers (Fig. 6B) measured by PSec was 7.02�10�3 cm/min, 5.84�10�3 cm/min and 5.35�10�3 cm/min for DGL/DNA,DGLePEG/DNA and DGLePEGeLeptin30/DNA NPs respectively,whichwas close to that reported by Pardridge [35]. The above resultsverified the integrity of BCECs monolayer during the experiments.

3.7. In vitro gene transfection

The transfection efficiency mediated by DGL series vector/DNANPs was assessed in BV-2 cells. Fig. 7 gave a qualitative comparisonof the DGL series vector/DNA NPs as well as positive control lip-ofectamine2000. The RFP expression of DGLePEGeLeptin30/DNAand lipofectamine2000 was much higher that of DGL/DNA andDGLePEG/DNA NPs. The luciferase activity of DGLePEGeLeptin30/DNA and lipofectamine2000 was 4-fold higher than that of DGL/DNA and DGLePEG/DNA NPs.

3.8. In vitro cytotoxicity

The cytotoxicity of the three DGL series vector/DNA NPs atdifferent concentrations was evaluated in BCECs and BV-2 cells byMTT assay. Lipofectamine2000 was used as positive control. Asshown in Fig. 9A, the cytotoxicity of the three NPs at low, medium,high concentration in BCECs was similar to that of lipofect-amine2000 which was thought to be safe enough in cells. Mean-while the cell viability in BV-2 cells at both 2 h and 48 h showed nosignificant difference from that of lipofectamine2000 (Fig. 9B andC). Fig. 9B revealed that the PEGylation and/or brain-targetingligand modification had improved the cell viability slightly.

3.9. In vivo imaging of mice administrated NPs

Nude mice were injected with the EMA-labeledDGLePEGeLeptin30/DNA NPs, and DGL/DNA NPs as control. In vivofluorescent images were taken at 80 min after injection. As shown

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Fig. 4. Cellular uptake of DGL/DNA (A, D, G, J, M, P), DGL-PEG/DNA (B, E, H, K, N, Q) and DGL-PEG-Leptin30/DNA (C, F, I, L, O, R) NPs at 4 �C (DeF) and with different inhibitors (GeR)was examined by fluorescent microscopy after a 60-min incubation. BCECs were treated with different inhibitors including free leptin30 peptide (GeI), transferrin (JeL), filipin(MeO) and colchicine (PeR). AeC was control without any inhibition. Red: EMA-labeled DNA. Original magnification: �200.

Y. Liu et al. / Biomaterials 31 (2010) 5246e5257 5251

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Fig. 5. Scatchard analysis. (main graph) The saturation curve showed that the binding of 125I-DGL-PEG-Leptin30 to BCECs is saturable in both low (A) and high (B) concentrationranges. (inset) Scatchard analysis of the binding data showed the presence of two independent classes of binding sites. Nonlinear regression analysis was used to estimate thedissociation constant (Kd) and binding capacity (Bmax) of the high-affinity site (A) and the low-affinity site (B), respectively.

Y. Liu et al. / Biomaterials 31 (2010) 5246e52575252

in Fig. 10, EMA-labeled DNAwas obviously accumulated in brain inthe mice treated with the DGLePEGeLeptin30/DNA NPs (Fig. 10B).While, the fluorescence in the brain of the DGL/DNA NPs treatedmice was not so significant (Fig. 10A). Fig. 10 inset shows that thebrain uptake of DGLePEGeleptin30/DNA NPs (right) was relativelyhigher than that of DGL/DNA NPs (left).

3.10. Qualitative analysis of distribution of gene expression in themouse brain

RFP expression in the cortical layer, hippocampus, caudateputamen and substantia nigra at 48 h after administrated DGL/DNA, DGLePEG/DNA, or DGLePEGeleptin30/DNA NPs were shownin Fig. 11AeL. The RFP expression in the four regions had nearly nodifference between DGL/DNA and DGLePEG/DNA NPs (Fig. 11AeH).For the DGLePEGeleptin30/DNA NPs, gene expression wasobserved in all the four regions (Fig. 11IeL) and was much higher

than that of the DGL/DNA and DGLePEG/DNA NPs, especially inhippocampus and substantia nigra.

3.11. In vivo gene quantitative expression

The transfection efficiencies of DGL/pGL2, DGLePEG/pGL2, andDGLePEGeLeptin30/pGL2 NPs in the principal organs weremeasured after 48 h (Fig. 12). The luciferase expression of theDGLePEGeLeptin30/pGL2 NPs in the brain was 1160 units/mgprotein, about 1-fold higher than that of the DGL/pGL2 NPs(639 units/mg protein) and DGLePEG/pGL2 NPs (715 units/mgprotein) (Fig. 12A).

The luciferase expression of DGL/DNA NPs was higher in heartand lower in kidney than that of DGLePEG/DNA,DGLePEGeLeptin30/DNANPs. In spleen, the luciferase expression issimilar for DGL/DNA and DGLePEG/DNA NPs, whereas the expres-sion levels of liver and lung were not changed markedly (Fig. 12B).

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Fig. 6. (A) The permeability of 125I-labeled NPs across BCECs monolayers at variousincubation times. Results are reported as mean� S.E.M (n¼ 4). *p< 0.05; **p< 0.01;***p< 0.001, significance represents DGL-PEG-Leptin30/DNA NPs vs. other two groups.(B) 14C-sucrose volume cleared across BCECs monolayers in continuous culture plottedvs. time of transport at 37 �C with NPs. Results are reported as mean� S.E.M (n¼ 4).

Fig. 7. The qualitative evaluation of gene expression in vitro. The fluorescence images of RFPDNA (B), DGL-PEG-Leptin30/DNA NPs (C) and Lipofectamine2000/DNA (D) NPs. Red: RFP. O

Fig. 8. The quantitative evaluation of gene expression in vitro. Luciferase activity wasmeasured 48 h post-transfection and expressed as light units per mg pro. ***p< 0.001,compared with the DGL/DNA and DGL-PEG/DNA NPs. Data represent the mean� S.E.M(n¼ 6).

Y. Liu et al. / Biomaterials 31 (2010) 5246e5257 5253

4. Discussion

Synthetic dendrimers have been successfully applied as non-viral gene vectors. Meanwhile, PEGylation and targeting ligandmodification render them better biocompatibility and tissue-selectivity. Diagnostic and therapeutic gene could be delivered intobrain by the specific binding between brain-targeting ligands andreceptors on BBBmediated transcytosis pathway. As a result, brain-targeting gene delivery systems based on dendrimers especially

expression in BV-2 cells were taken 48 h post-infection with DGL/DNA (A), DGL-PEG/riginal magnification: �200.

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Fig. 9. Cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay performed 2 h in BCECs (A) and BV-2 cells (B) or 48 h in BV-2 cells (C)after transfection of DGL/DNA, DGL-PEG/DNA, DGL-PEG-leptin30/DNA NPs at different concentrations compared with lipofectamine2000 (n¼ 3).

Y. Liu et al. / Biomaterials 31 (2010) 5246e52575254

that could be degraded in vivo hold great promise for the furtherexperimental or clinical applications.

The purpose of this work was to evaluate the brain-targetingefficiency of the leptin30 modified gene delivery system. The

DGLePEGeLeptin30 was proved to be synthesized successfully(Fig. 1) and complexed with plasmid DNA by electrostatic interac-tions (Fig. 2), yielding nano-scaled, positively charged nano-particles (NPs).

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Fig. 10. In vivo distribution of NPs after i.v. administration. Images were taken at 80 min after injection. (A) was control with DGL/DNA NPs injection and (B) was injected with DGL-PEG-leptin30/DNA NPs. The two images in the inset shows the relative brain accumulation of DGL/DNA NPs (left) and DGL-PEG-leptin30/DNA NPs (right).

Y. Liu et al. / Biomaterials 31 (2010) 5246e5257 5255

Three factors might influence the cellular uptake ofDGLePEGeLeptin30/DNA NPs in BCECs, namely surface charge,PEGylation and brain-targeting ligand leptin30 modification. Thecellular uptake efficiency increased with the degree of PEGylationand brain-targeting ligand modification on theDGLePEGeLeptin30/DNA NPs from none to median (Fig. 3AeC).However, the uptake efficiency changed slightly between the NPswith median and high (Fig. 3C and D) modification of the PEGyla-tion and brain-targeting ligand. It was possible that no significantdifference in uptake between the median and high brain-targetingmodification NPs. The similar results were observed in a folate-decorated drug delivery system [34].

Fig. 11. The qualitative evaluation of gene expression in vivo. Distribution of gene expressionDGL-PEG-leptin30/DNA NPs (IeL) 2 days after i.v. administration. Frozen sections (20 mm thcortical layer (D, H, L) were examined by fluorescent microscopy. The sections were stained wmagnification: �100.

The cellular uptake of all the three NPs could be inhibited at 4 �C(Fig. 4DeF) indicating the endocytosis was an energy-dependentprocess. The endocytosis mechanism of NPs was preliminaryinvestigated. The uptake of DGLePEGeLeptin30/DNA NPs wasdependent with leptin30 specifically and inhibited by the excessfree leptin30, which was different from the other two NPs (Fig. 4).And the Kd of DGLePEGeLeptin30 binding to BCECs determined inthis study (Fig. 5) was lower than of leptin reported by Pardridge[35]. In addition, the maximum binding of DGLePEGeLeptin30enhanced compared with that of leptin. This difference may attri-bute to the adsorptive effect between positively charged DGL andnegatively charged cell membrane (Fig. 5). It may also be due to the

in brains of ICR mice treated with DGL/DNA NPs (AeD), DGL-PEG/DNA NPs (EeH), andick) of caudate putamen (A, E, I), hippocampus (B, F, J), substantia nigra (C, G, K) andith 300 nM DAPI for 10 min at room temperature. Green: GFP. Blue: cell nuclei. Original

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Fig. 12. The quantitative evaluation of gene expression in vivo. Luciferase expression48 h after i.v. administration of DGL/DNA, DGL-PEG/DNA, and DGL-PEG-Leptin30/DNANPs in ICR mice at a dose of 50 mg DNA/mouse. Luciferase expression of brain (A) andother solid organs (B) is plotted as light units per mg protein. *p< 0.05 compared withthe DGL/DNA and DGL-PEG/DNA NPs. Data are expressed as mean� S.E.M (n¼ 4).

Y. Liu et al. / Biomaterials 31 (2010) 5246e52575256

short peptide derived from longer peptide exhibits higher receptoraffinity than the original peptides [36].

The above evidence suggested the combined cellular internali-zation promoting effect by the DGL polymer and leptin30 ligand.The cellular uptake of DGLePEGeLeptin30/DNA NPs could beinhibited by transferrinwhich was thought to be the typical markerbeing internalized through clathrin-dependent pathway [37]. Theresults were in good agreement with the previous study whichreported leptin receptor was internalized by clathrin-dependentpathway [38,39] and confirmed leptin receptor-related endocy-tosis. Filipin acts as an inhibitor of caveolae mediated uptake bybinding cholesterol, an important component for caveolae forma-tion [40]. The cellular internalization appears to be also mediatedthrough caveolae which is involved in the adsorption-mediatedendocytosis process [41]. Meanwhile, colchicine is used in thisstudy as macropinocytosis inhibitor [42]. The uptake of DGL/DNANPs was mediated by caveolae mediated endocytosis and macro-pinocytosis. The similar concepts were mentioned as macro-pinocytosis could also mediate the uptake of cationic carriers [43].DGLePEGeLeptin30/DNA NPs endocytosis had relation to all thethree endocytosis pathway. It confirmed that the ligand and thepolymer both contribute to the cellular internalization of the NPs.In addition, the confocal analysis of BCECs incubated with DGL/DNANPs indicated that EMA-labeled NPs could escape from endosomeseffectively at 1 h after incubation (data not shown).

DGLePEGeLeptin30/DNA NPs could be transported across invitro BBB model consisted of BCECs monolayers more effectively

than DGLePEG/DNA and DGL/DNA NPs as the permeability ofDGLePEGeLeptin30/DNA NPs was significantly higher than that ofDGLePEG/DNA NPs and DGL/DNANPs from 10 min after incubation(Fig. 6A). While transendothelial electrical resistance (TEER) andpermeability of 14C-sucrose (Fig. 6B) demonstrated that the integ-rity remained during the whole incubation process [35]. It could bededuced the specific binding between leptin30 and leptin receptorsplayed an active role in the transcytosis of NPs.

In vitro transfection efficiency was evaluated in BV-2 microgliacells. BV-2 cells as well as many other brain cells were reported toexpressed the long (OBRl) and short (OBRs) isoforms of the leptinreceptors [29]. Both qualitative (Fig. 7) and quantitative (Fig. 8)results revealed the transfection ability of DGLePEGeLeptin30/DNA NPs was higher than that of DGL/DNA and DGLePEG/DNA NPswhile equal to that of lipofectamine2000. It could be also explainedthat leptin30 could bind leptin receptors on BV-2 cells and beinternalized by receptor mediated endocytosis. The DNA accumu-lation of DGLePEGeLeptin30/DNA NPs was found higher than thatof DGL/DNA NPs in brain after 80 min (Fig. 10). The report geneexpression at different regions in brain (cortical layer, hippo-campus, caudate putamen and substantia nigra) has clearlyrevealed the preferential localization of DGLePEGeLeptin30/DNANPs in the brain when compared with other two controls (Fig. 11).The transfection efficiency in vivo was in accordance to the resultsin BV-2 cells. A relative high report gene expression also showedqualitatively in brain (Fig. 12A). The in vivo transfection abilitydeclined slightly compared with the in vitro results (Figs. 7 and 8).The reasons may be the specific binding between targetingDGLePEGeLeptin30/DNA NPs and leptin receptors on BBB wasinterfered by endogenic leptin. It is reported the plasma level ofleptin in normal mice is about 280 nM [44] and doesn’t changeacutely with food administration [45], while the plasma level ofleptin30 modified NPs was estimated no more than 5000 nM in thisstudy. The targeting modification of NPs was sufficient enough toexert their targeting effects. This result was in agreement with thecellular uptake comparison result which demonstrated the uptakeefficiency altered slightly as the targeting modification increasingfrom median to high. The accumulation in heart may be caused bythe property of DGL but not relate to the modification of Leptin30since the report gene expressions were higher in DGL/DNA NPsadministration group than that in DGLePEG/DNA andDGLePEGeLeptin30/DNA NPs administration groups (Fig. 12B). Thesimilar results were observed in gene delivery systems based onPAMAM with different modification [12,13]. It is worth optimizingthe charge ratio N/P to achieve the best in vivo transfection effi-ciency further.

The cytotoxicity of the three series vector/DNA NPs at 2 h and48 h in BCECs and BV-2 cells was relatively low at all the concen-trations tested, while lipofectamine2000 didn’t show much cyto-toxicity in this experiment (Fig. 9). Compared with some syntheticpolymers, DGL was thought to be less toxic and biodegradable [17],hence possess better biocompatibility due to the peptide-likelinkage between the monomers. And the accumulative or long-term toxicity was relatively low. The above results further indicatethe potent brain-targeting ability of leptin30 modified PEGylatedDGL as the efficient gene vector.

5. Conclusions

DGLePEGeLeptin30 was synthesized and complexed withplasmid DNA yielding nanoparticles. The mechanism ofDGLePEGeLeptin30/DNA NPs mediated brain-targeting is affectedby surface charge, PEGylation and leptin30 modification and couldbe characterized as a clathrin and caveolae mediated energy-depending endocytosis. Compared with in vitro transfection results

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Y. Liu et al. / Biomaterials 31 (2010) 5246e5257 5257

in vivo transfection ability may be interfered by endogenic leptinslightly, the DGLePEGeLeptin30/DNA NPs were demonstrated tobe efficient enough for brain-targeting gene delivery. It could bebelieved that DGLePEGeLeptin30 holds great promise as the non-viral vector for relatively safe and efficient brain-targeting genedelivery.

Acknowledgements

The authors thank Pro. Jianhua Zhu (School of Pharmacy,Fudan University) for the 125I-labeled work. This work was sup-ported by the grant from National Basic Research Program(2007CB935802) of China (973program), National Natural Foun-dation of China (30973652) and “Key new drug creation program”

2009ZX09310-006.

Appendix

Figures with essential colour discrimination. Certain figures inthis article, in particular Figs. 3,4,7,10 and 11 are difficult to interpretin black and white. The full colour images can be found in theonline version, at doi:10.1016/j.biomaterials.2010.03.011.

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