A leptin derived 30-amino-acid peptide modified pegylated

Biomaterials 31 (2010) 5246e5257
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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
A leptin derived 30-amino-acid peptide modified 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
b
Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China
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
a b s t r a c t
Article history:
Received 11 January 2010
Accepted 4 March 2010
Available online 10 April 2010
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. Specific 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 efficiency after intravenous injection. Dendrigraft polyL-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 efficiency 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.
Keywords:
Brain targeting
Gene delivery
Leptin
Non-viral vector
Dendrigraft poly-L-lysine
1. Introduction
Central nervous system diseases such as Alzheimer’s disease
(AD), Parkinson’s 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
efficient 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
modification. Despite viral gene vectors have been proved to be
efficient 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 intensively investigated in gene delivery system [9e11]. PAMAM have
been successfully applied to non-viral gene delivery systems in our
* Corresponding author. Tel./fax: þ86 21 5198 0079.
E-mail address: [email protected] (C. Jiang).
0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2010.03.011
previous studies [12e14]. The critical concerns about the synthetic
polymers include safety and efficiency. 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 modified 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 wellsuitable for the development of drug delivery vehicles [21].
However, because of lack of selectivity, the cell-penetrating peptide
modified 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 modified with ligands such
as transferrin and lactoferricin, could be uptaken by brain capillary
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
endothelial cells (BCECs) and cross BBB via endocytosis pathway
mediated by the specific ligand-receptor binding [12,22]. Compared
with the above large molecule protein ligands, short peptide ligands
have the advantages of a relatively small molecular weights, being
easily synthesized, relatively low cytotoxicity and immunogenicity,
and degradability [18]. There have been quite a few research papers
about the short peptide ligands modified drug delivery systems
recently. And we have previously reported a 29 amino acids peptide
derived from rabies virus glycoprotein could serve as brain-targeting ligand inducing efficient brain-targeting gene delivery [13]. On
the basis of the above evidence, a 30 amino acids peptide derived
from leptin was investigated in this research.
Leptin, a 146 amino acid polypeptide secreted into the bloodstream by adipocytes, acts centrally on leptin receptor-expressing
cells located in the hypothalamus and other parts of the brain,
leading to decreased food intake and retarding weight gain [23,24]. It
has been widely reported that leptin could be taken up by the brain
[25,26]. Considering the 16 kDa molecular weight of leptin preclude
the possibility of passive diffusion across the bloodebrain barrier,
a process that provides limited entry for some relatively small
molecules, the leptin receptor mediated transport is believed to be
one of the possible mechanisms besides leptin receptor independent
pathway. Barrett’s group has identified several leptin derived
peptides that are taken up by the brain [27]. Among these, peptide
corresponded to position 61e90 (leptin30, 30 amino acids) was
observed to possess the highest brain: plasma ratio which was
equivalent to that of leptin. For leptin30, the majority of the radioactivity was localized more to parenchyma than capillaries. As the
regions conferring brain uptake correspond closely to the sequences
that are necessary for receptor binding, the results reinforce the view
that 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 ligand
modified on the surface of gene vectors. Furthermore, it could be
deduced that after transcytosis across the BBB, leptin30 could
enhance internalization to the brain parenchyma cells by the
specific ligand-receptor mediated endocytosic pathway. Therefore,
leptin30 modified gene delivery system may have significant
repercussions for brain diseases therapeutics.
In this study, leptin30 was modified by covalent linkage bond on
the surface of DGL through bifunctional polyethyleneglycol (PEG),
yielding DGLePEGeleptin30. The brain-targeting efficiency of
DGLePEGeleptin30 as gene delivery vectors was evaluated in vitro
and in vivo. Furthermore, the uptake mechanism of
DGLePEGeLeptin30/DNA NPs was explored.
2. Materials and methods
2.1. Materials
DGL were purchased from Colcom, France. a-Malemidyl-u-N-hydroxysuccinimidyl polyethyleneglycol (NHS-PEG-MAL, MW 3400) was obtained from
Nektar Therapeutics (Huntsville, AL, USA). The peptide leptin30 with a cysteine on Cterminal (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 purified
using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). Filipin, colchicine
and were purchased from SigmaeAldrich (St. Louis, MO, USA). EMA and 4,6-diamidino-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 purchased
from the Sino-British SiPPR/BK Lab. Animal Ltd and 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.
2.2. Cell culture
The primary cultured brain capillary endothelial cells (BCECs) isolated from
BALB/C mouse were kindly provided by Prof. J. N. Lou (the Clinical Medicine
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Research Institute of the ChineseeJapanese Friendship Hospital). Primary BCECs
were cultured as described previously [30]. Briefly, BCECs were expanded and
maintained in special Dulbecco’s modified Eagle medium (DMEM) (SigmaeAldrich)
supplemented with 20% heat-inactivated fetal calf serum (FCS), 100 mg/ml epidermal
cell 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 humidified
atmosphere containing 5% CO2. All cells used in this study were between passage 15
and passage 30.
BV-2 microglia cells were gifts from Prof. L.Y. Feng (Shanghai Institute of
material medica, Chinese academy of sciences). BV-2 cells were cultured in DMEM
(high sucrose), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin
and 100 mg/ml streptomycin at 37 C in a humidified 5% CO2 incubator. All cells used
in 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 (pH
8.0) for 2 h at room temperature. The primary amino groups on the surface of DGL
were specifically reacted with the NHS groups of the bifunctional PEG derivative.
The resulting conjugate, DGLePEG, was purified by ultrafiltration through
a membrane (cutoff 5 kDa) and the buffer was changed to PBS (pH 7.0). Then
DGLePEG was reacted with peptide leptin30 in PBS (pH 7.0) for 24 h at room
temperature. The MAL groups of DGLePEG were specifically reacted with the thiol
groups of Leptin30. Ethidium monoazide (EMA) is a fluorescent photoaffinity label
that, after photolysis, binds covalently to plasmid DNA. Thus, after complexing with
the EMA-labeled plasmid DNA, the DGL series NPs was fluorescent. For synthesis of
EMA-labeled DNA, EMA was labeled on DNA through UV exposure 1 h and series
purification.
2.4. Characterization of DGLePEGeLeptin30
The characteristics of DGLePEGeLeptin30 were analyzed by nuclear magnetic
resonance (NMR) spectroscopy. Basically, DGLePEGeLeptin30 was freeze-dried,
solubilized in D2O and analyzed in a 400 MHz spectrometer (Varian, Palo Alto, CA, USA).
2.5. Preparation and characterization of dendrimers/DNA nanoparticles(NPs)
Dendrimers (DGL, DGLePEG, and DGLePEGeLeptin30) were freshly prepared
and diluted to appropriate concentrations in PBS (pH 7.4). DNA solution (100 mg
DNA/ml 50 mM sodium sulfate solution) was added to obtain specified weight
ratios (6:1, DGL to DNA, w/w) and immediately vortexed for 30 s at room
temperature. Freshly prepared NPs were used in the experiments that follow. The
mean diameter and zeta-potential of DGL series vectors (DGL, DGLePEG,
DGLePEGeLeptin30)/DNA NPs were determined by dynamic light scattering
using a Zeta-Potential/Particle Sizer NicompÔ 380 ZLS (PSS Nicomp Particle Size
System, 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/
DNA were freshly prepared with DGL to DNA at weight ratio of 6:1. 0.7% agarose gel
electrophoresis was performed to evaluate the DNA encapsulation effect caused by
DGL in the NPs compared with naked DNA. To determine the DNase I stability, DNase
I (Takara Bio, Japan) (1 U/mg of DNA) was added to each NPs, and the mixtures were
incubated at 37 C for 30 min. 250 mM EDTA solution was added and incubated at
room temperature for 10 min to inactivate the DNase I. After that, 15 mg/ml sodium
heparin was added and incubated at room temperature for 2 h to release the DNA
from the NPs. All the samples were analyzed by 0.7% agarose gel electrophoresis. The
integrity of the plasmid in each sample was compared with untreated naked DNA
and DNase I treated naked DNA.
2.7. Cellular uptake of dendrimers/DNA NPs and with different endocytosis
inhibitors
BCECs were seeded at a density of 2 104 cells/well in 24-well plates (CorningCoaster, Tokyo, Japan), incubated for 72 h, and checked under the microscope for
similar confluency and morphology. After this, BCECs were incubated with EMAlabeled DGLePEG/DNA or DGLePEGeLeptin30/DNA NPs at the concentration of
20 mg/well measured by DGL in the present of serum-free medium for 1 h at 37 C. In
case of inhibitors groups, different inhibitors were pre-incubated with cells 10 min,
following the addition of the DGLePEGeLeptin30/DNA NPs treated at the condition
mentioned 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, incubated
for 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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Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
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 were
removed and washed with cold PBS 7.4 for three times. 200 ml 1 M NaOH were added
to lysis cells for 3 h. 100 ml 2 M HCl was used to neutralize NaOH. The radioactivity of
the final well contents was analyzed by a g-counter (SN-695, China). The above
experiments were performed to determine the total binding of vector with leptin
receptors. Nonspecific binding was determined by adding 5 mM nonradioactive
DGLePEGeLeptin30 to each reaction mixture in a parallel assay. The amount of
cellular 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-well
Transwell filters of 1.0 mm mean pore size, 0.33 cm2 surface area (FALCON Cell
Culture Insert, Becton Dickinson Labware, U.S.A.). After about 3 days’ culture, the
cells were checked under the microscope for complete confluency. The cell monolayer integrity was monitored using an epithelial voltohmmeter (MILLICELLÒ-ERS,
Millipore, U.S.A.) to measure the transendothelial electrical resistance (TEER). Only
cell monolayers with TEER exceeding 200 U cm2 were selected for this experiment
[32].
Radiolabeled DGL was applied to synthesize DGLePEG/DNA and
DGLePEGeLeptin30/DNA NPs. 300 ml fetal bovine serum (FBS)-free medium containing 0.6 mCi 14C-sucrose was added to the donor chamber at time 0, to monitor the
integrity of BCECs monolayers. At the same time, BCECs monolayers were treated
with 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 at
50 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 of
scintillation 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. The
apparent permeability (Papp) was calculated according to Irvine et al. [33] as follows:
Papp ¼ ðdQ =dtÞ ð1=C0 Þ ð1=AÞ
where 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 measured
from time 0e60 min to monitor the integrity of monolayers [35]. Basically, the
cleared volume of 14C-sucrose was plotted vs. time, and the slope was estimated by
linear regression analysis to give the mean 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 BCECs monolayer and the filter, PSf is the PS value for
the 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 in
a DMEM medium containing 10% FBS, and grown to reach 70e80% confluence prior
to transfection. Before transfection, the medium was exchanged with fresh serumfree medium. The cells were treated with different NPs solutions containing 5 mg of
plasmid DNA for 4 h at 37 C. After exchange with a fresh serum-containing
medium, cells were further incubated for 2 days after transfection. 5 mg plasmid DNA
mixed with Lipofectamine2000 according to the standard protocol as described in
instruction served as positive control. In the case of qualitative evaluation, the red
fluorescence images were taken using a fluorescence microscope. For luciferase
activity assay, medium was removed and the cells were rinsed with DPBS and
shaken for 30 min at room temperature in 150 ml of luciferase cell culture lysis
reagent supplied by the Promega Luciferase Assay Kit. The lysis solution was
centrifuged at 14,000g for 2 min at 4 C. Luciferase activity was measured by BPCL-1
Ultra-Weak CL and BL Analyzer and total amount of cellular protein was determined
by 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 were
seeded in a 96-well tissue culture plate at 5000 cells per well in 90 ml DMEM
medium containing 10% FBS. Cells achieving 70e80% confluence after 24 h were
exposed to 100 ml of different NPs solutions with various concentrations and Lipofectamine200 at equivalent to 0.2 mg DNA per well for 2 h. Then, 100 ml of stock
solution of MTT (1 mg/ml in Hank’s) was added to each well. After 3 h of incubation
at 37 C, each medium was removed and 100 ml of DMSO was added to each well to
dissolve the formazan crystals formed by proliferating cells. Cells without treatment
were used as a control. Absorbance was measured at 570 nm using a microplate
reader (BIO-TEK, MQX200R PowerWaveÔ XS). Cell viability of each group was
expressed as a percentage relative to that of control cells.
The same experiments were also performed on BV-2 cells. For 48 h assay, DMEM
was added after 2 h incubation with NPs.
2.12. In vivo imaging analysis
The DGLePEGeLeptin30/DNA NPs labeled by EMA were injected into the tail
vein 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 were
extirpated 2 h after injection for comparing the relative accumulation. EMA-labeled
DGL/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/pRFP
and DGLePEG/pRFP NPs were injected into the tail vein of mice at a dose of 50 mg
DNA/mouse. About 48 h later, animals were anesthetized with diethyl ether and
killed by decapitation. The brains were removed, fixed in 4% paraformaldehyde for
48 h, placed in 15% sucrose solution until subsidence (6 h), then in 30% sucrose
until 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 were
prepared with a cryotome Cryostat (Leica, CM 1900, Wetzlar, Germany) and
stained with 300 nM DAPI for 10 min at room temperature. After washing twice
with PBS (pH 7.4), the sections were immediately examined under the fluorescence
microscope.
2.14. In vivo gene quantitative expression
The DGLePEGeLeptin30/pGL2 NPs (6:1, DGL to DNA, w/w) were injected into
the tail vein of mice at a dose of 50 mg DNA/mouse. At 48 h after injection, the mice
were humanely decapitated and the principal organs (including brain, heart, liver,
spleen, lung, and kidney) were extirpated. The organs were carefully washed with
distilled water and homogenized in 1 ml lysis reagent (Promega, Madison, WI, USA)
using a JY92-IIN tissue homogenizer. The homogenate was centrifuged at 14,000g
for 20 min at 4 C. Luciferase activity and cellular proteins in the supernatant were
quantified by a Luciferase Assay System and total amount of cellular protein was
determined by a BCA Protein Assay, respectively. The results were expressed as light
units/mg protein. Meanwhile DGL/pGL2 and DGLePEG/pGL2 NPs were also injected
for comparison.
3. Results
3.1. Characterization of DGLePEGeLeptin30 and
DGLePEGeLeptin30/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 of
protons of leptin30 peptide have multiple peaks between 4.3 and
1.1 ppm. The NMR spectrum of DGLePEG had a characteristic peak
of the MAL group in PEG at 6.7 ppm (data not shown). The MAL peak
disappeared in the NMR spectrum of DGLePEGeleptin30, whereas
the repeat units of PEG still presented a sharp peak at 3.6 ppm
(Fig. 1) showing that the MAL group had reacted with the thiol group
of peptide leptin30. The NMR spectra result proved the existence of
the conjugate structure of DGLePEGeleptin30. Meanwhile Ellman’s
reagent was used to determine the percentage of unreacted thiol.
And little thiol was detected (data not shown) which could be
explained as the thiol at the end of leptin30 reacted with the MAL at
one end of PEG specifically. The particle size and zeta-potential were
measured. The results showed the vector/pDNA complex was nanoscaled (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
and
DGLePEGeLeptin30/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 and
DGLePEGeLeptin30/DNA) with DGL to DNA at weight ratio of 6:1
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
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Fig. 1. NMR spectra of DGL-PEG-leptin30 in D2O at 400 MHz.
could encapsulate DNA completely with no electrophoresis shift
(Fig. 2A lane 3e5) compared with naked DNA (Fig. 2A lane 2). To
investigate whether DGL series vectors could protect DNA against
enzymatic degradation, naked DNA and the three DGL series NPs
were incubated with DNase I. For all the three NPs, no electrophoresis shifts were observed after that (data not shown). To check the
integrity of plasmid DNA in NPs after DNase I incubation, sodium
heparin was added to release DNA. Fig. 2B shows that naked plasmid
DNA (lane 2) was completely digested by DNase I, whereas all the
three NPs (lane 3e5) could protect DNA from digestion.
3.3. Uptake comparison of DGL series vector/DNA NPs by BCECs in
vitro
The EMA-labeled DGL series vector/DNA NPs were used to
investigate cellular uptake characteristics. The results were shown
qualitatively using fluorescent images. DGL with different PEGylation and leptin30 brain-targeting modification were synthesized as
the gene vectors. The uptake efficiency was investigated in BCECs to
determine an appropriate proportion for the subsequent experiments. The fluorescence intensity exhibited by BCECs had slightly
increased when cells were treated with the EMA-labeled
DGLePEGeleptin30/DNA NPs at 1:5:2 (DGL:PEG:Leptin30, weight
ratio) and 1:10:3 (Fig. 3C and D) compared with DGL/DNA NPs and
DGLePEGeleptin30/DNA (1:2:1) (Fig. 3A and B). As the ratio 1:5:2
and 1:10:3 showed no significant difference in uptake, DGL:PEG:
Leptin30 at the weight ratio of 1:5:2 was selected and studied in
further 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/DNA
NPs declined dramatically at 4 C (Fig. 4DeF) compared with
treated at 37 C (Fig. 4AeC). Excessive free leptin30 could inhibit
the uptake of DGLePEGeleptin30/DNA NPs (Fig. 4G), but had little
influence on that of non-leptin30 modified NPs (Fig. 4H and I).
Different endocytosic pathway inhibitors were used to clarify the
endocytosis process. Treatment with transferrin resulted in
a decrease of fluorescent intensity (Fig. 4K and L) of the DGLePEG/
DNA and DGLePEGeLeptin30/DNA NPs uptake while showed no
Fig. 2. A: Agarose gel electrophoresis was performed. Lane 1: marker; lane 2: naked DNA; lane 3: DGL/DNA NPs; lane 4: DGL-PEG/DNA NPs; lane 5: DGL-PEG-Leptin30/DNA NPs. B:
The stability of DNA-loaded NPs against DNase I digestion. Plasmid DNA was released from the NPs by the addition of sodium heparin separated by agarose gel electrophoresis after
DNase I incubation. Lane 1: naked plasmid DNA; lane 2: naked plasmid DNA treated with DNase I; lane 3e5: DGL/DNA, DGL-PEG/DNA, and DGL-PEG-leptin30/DNA NPs with
treatment of heparin after DNase I incubation.
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Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
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: DGLPEG-Leptin30 (weight ratio ¼ 1:2:1)/DNA NPs. C: DGL-PEG-Leptin30 (weight ratio ¼ 1:5:2)/DNA NPs. D: DGL-PEG-Leptin30 (weight ratio ¼ 1:10:3)/DNA NPs. Red: EMA-labeled
DNA. Original magnification: 200.
effect on the uptake of DGL/DNA NPs (Fig. 4J). And the fluorescent
intensity of DGL/DNA and DGLePEGeLeptin30/DNA NPs declined
to some extent (Fig. 4M and O) by filipin. The treatment of colchicine seemed to have little influence on the DGLePEG/DNA NPs
uptake (Fig. 4Q). However, the fluorescent intensity of DGL/DNA
and DGLePEGeLeptin30/DNA NPs decreased with the pre-incubation of colchicine (Fig. 4P and R).
BCECs monolayers (Fig. 6B) measured by PSec was 7.02 103 cm/
min, 5.84 103 cm/min and 5.35 103 cm/min for DGL/DNA,
DGLePEG/DNA and DGLePEGeLeptin30/DNA NPs respectively,
which was close to that reported by Pardridge [35]. The above results
verified the integrity of BCECs monolayer during the experiments.
3.5. Scatchard analysis of leptin receptor-binding assay
The transfection efficiency mediated by DGL series vector/DNA
NPs was assessed in BV-2 cells. Fig. 7 gave a qualitative comparison
of the DGL series vector/DNA NPs as well as positive control lipofectamine2000. The RFP expression of DGLePEGeLeptin30/DNA
and lipofectamine2000 was much higher that of DGL/DNA and
DGLePEG/DNA NPs. The luciferase activity of DGLePEGeLeptin30/
DNA and lipofectamine2000 was 4-fold higher than that of DGL/
DNA and DGLePEG/DNA NPs.
Binding of 125I-DGLePEGeLeptin30 to BCECs was saturable in
the presence of excess of nonradiolabeled DGLePEGeLeptin30 in
both 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 binding
sites. The parameters governing the binding of DGLePEGeLeptin30
to the BCECs, estimated by nonlinear regression analysis, indicated
a high-affinity binding site (Fig. 5, inset A) with a dissociation
constant K1d of 1.737 0.21 nM (estimate SD) compared with that
of leptin (5.1 2.8 nM reported by Pardridge [25]) and binding
maximum (B1max) of 0.6258 0.02 pmol/mg pro. (compared with
leptin, 0.34 0.16 pmol/mg pro. reported). While, low-affinity and
high-capacity class of binding sites (Fig. 5, inset B) had an estimated
dissociation constant (K2d) of 421.8 66.13 nM (leptin, 743 226 nM
reported) and binding maximum (B2max) 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 and
DGLePEGeLeptin30/DNA NPs were shown in Fig. 6A. Papp of
DGLePEGeLeptin30/DNA NPs was significantly higher than that of
DGL/DNA and DGLePEG/DNA NPs after 10 min. The transendothelial
electrical resistance (TEER) showed no significant difference from
that of controls (data not shown). The permeability of 14C-sucrose of
3.7. In vitro gene transfection
3.8. In vitro cytotoxicity
The cytotoxicity of the three DGL series vector/DNA NPs at
different concentrations was evaluated in BCECs and BV-2 cells by
MTT assay. Lipofectamine2000 was used as positive control. As
shown in Fig. 9A, the cytotoxicity of the three NPs at low, medium,
high concentration in BCECs was similar to that of lipofectamine2000 which was thought to be safe enough in cells. Meanwhile the cell viability in BV-2 cells at both 2 h and 48 h showed no
significant difference from that of lipofectamine2000 (Fig. 9B and
C). Fig. 9B revealed that the PEGylation and/or brain-targeting
ligand modification had improved the cell viability slightly.
3.9. In vivo imaging of mice administrated NPs
Nude
mice
were
injected
with
the
EMA-labeled
DGLePEGeLeptin30/DNA NPs, and DGL/DNA NPs as control. In vivo
fluorescent images were taken at 80 min after injection. As shown
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
5251
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.
5252
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
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) concentration
ranges. (inset) Scatchard analysis of the binding data showed the presence of two independent classes of binding sites. Nonlinear regression analysis was used to estimate the
dissociation constant (Kd) and binding capacity (Bmax) of the high-affinity site (A) and the low-affinity site (B), respectively.
in Fig. 10, EMA-labeled DNA was obviously accumulated in brain in
the mice treated with the DGLePEGeLeptin30/DNA NPs (Fig. 10B).
While, the fluorescence in the brain of the DGL/DNA NPs treated
mice was not so significant (Fig. 10A). Fig. 10 inset shows that the
brain uptake of DGLePEGeleptin30/DNA NPs (right) was relatively
higher than that of DGL/DNA NPs (left).
3.10. Qualitative analysis of distribution of gene expression in the
mouse brain
RFP expression in the cortical layer, hippocampus, caudate
putamen and substantia nigra at 48 h after administrated DGL/
DNA, DGLePEG/DNA, or DGLePEGeleptin30/DNA NPs were shown
in Fig. 11AeL. The RFP expression in the four regions had nearly no
difference between DGL/DNA and DGLePEG/DNA NPs (Fig. 11AeH).
For the DGLePEGeleptin30/DNA NPs, gene expression was
observed in all the four regions (Fig. 11IeL) and was much higher
than that of the DGL/DNA and DGLePEG/DNA NPs, especially in
hippocampus and substantia nigra.
3.11. In vivo gene quantitative expression
The transfection efficiencies of DGL/pGL2, DGLePEG/pGL2, and
DGLePEGeLeptin30/pGL2 NPs in the principal organs were
measured after 48 h (Fig. 12). The luciferase expression of the
DGLePEGeLeptin30/pGL2 NPs in the brain was 1160 units/mg
protein, about 1-fold higher than that of the DGL/pGL2 NPs
(639 units/mg protein) and DGLePEG/pGL2 NPs (715 units/mg
protein) (Fig. 12A).
The luciferase expression of DGL/DNA NPs was higher in heart
and
lower
in
kidney
than
that
of
DGLePEG/DNA,
DGLePEGeLeptin30/DNA NPs. In spleen, the luciferase expression is
similar for DGL/DNA and DGLePEG/DNA NPs, whereas the expression levels of liver and lung were not changed markedly (Fig. 12B).
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
5253
Fig. 8. The quantitative evaluation of gene expression in vitro. Luciferase activity was
measured 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
(n ¼ 6).
4. Discussion
125
Fig. 6. (A) The permeability of
I-labeled NPs across BCECs monolayers at various
incubation times. Results are reported as mean S.E.M (n ¼ 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 plotted
vs. time of transport at 37 C with NPs. Results are reported as mean S.E.M (n ¼ 4).
Synthetic dendrimers have been successfully applied as nonviral gene vectors. Meanwhile, PEGylation and targeting ligand
modification render them better biocompatibility and tissueselectivity. Diagnostic and therapeutic gene could be delivered into
brain by the specific binding between brain-targeting ligands and
receptors on BBB mediated transcytosis pathway. As a result, braintargeting gene delivery systems based on dendrimers especially
Fig. 7. The qualitative evaluation of gene expression in vitro. The fluorescence images of RFP expression in BV-2 cells were taken 48 h post-infection with DGL/DNA (A), DGL-PEG/
DNA (B), DGL-PEG-Leptin30/DNA NPs (C) and Lipofectamine2000/DNA (D) NPs. Red: RFP. Original magnification: 200.
5254
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
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 (n ¼ 3).
that could be degraded in vivo hold great promise for the further
experimental or clinical applications.
The purpose of this work was to evaluate the brain-targeting
efficiency of the leptin30 modified gene delivery system. The
DGLePEGeLeptin30 was proved to be synthesized successfully
(Fig. 1) and complexed with plasmid DNA by electrostatic interactions (Fig. 2), yielding nano-scaled, positively charged nanoparticles (NPs).
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
5255
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 DGLPEG-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).
Three factors might influence the cellular uptake of
DGLePEGeLeptin30/DNA NPs in BCECs, namely surface charge,
PEGylation and brain-targeting ligand leptin30 modification. The
cellular uptake efficiency increased with the degree of PEGylation
and
brain-targeting
ligand
modification
on
the
DGLePEGeLeptin30/DNA NPs from none to median (Fig. 3AeC).
However, the uptake efficiency changed slightly between the NPs
with median and high (Fig. 3C and D) modification of the PEGylation and brain-targeting ligand. It was possible that no significant
difference in uptake between the median and high brain-targeting
modification NPs. The similar results were observed in a folatedecorated drug delivery system [34].
The cellular uptake of all the three NPs could be inhibited at 4 C
(Fig. 4DeF) indicating the endocytosis was an energy-dependent
process. The endocytosis mechanism of NPs was preliminary
investigated. The uptake of DGLePEGeLeptin30/DNA NPs was
dependent with leptin30 specifically and inhibited by the excess
free leptin30, which was different from the other two NPs (Fig. 4).
And the Kd of DGLePEGeLeptin30 binding to BCECs determined in
this study (Fig. 5) was lower than of leptin reported by Pardridge
[35]. In addition, the maximum binding of DGLePEGeLeptin30
enhanced compared with that of leptin. This difference may attribute to the adsorptive effect between positively charged DGL and
negatively charged cell membrane (Fig. 5). It may also be due to the
Fig. 11. The qualitative evaluation of gene expression in vivo. Distribution of gene expression in brains of ICR mice treated with DGL/DNA NPs (AeD), DGL-PEG/DNA NPs (EeH), and
DGL-PEG-leptin30/DNA NPs (IeL) 2 days after i.v. administration. Frozen sections (20 mm thick) of caudate putamen (A, E, I), hippocampus (B, F, J), substantia nigra (C, G, K) and
cortical layer (D, H, L) 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: 100.
5256
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
Fig. 12. The quantitative evaluation of gene expression in vivo. Luciferase expression
48 h after i.v. administration of DGL/DNA, DGL-PEG/DNA, and DGL-PEG-Leptin30/DNA
NPs in ICR mice at a dose of 50 mg DNA/mouse. Luciferase expression of brain (A) and
other solid organs (B) is plotted as light units per mg protein. *p < 0.05 compared with
the DGL/DNA and DGL-PEG/DNA NPs. Data are expressed as mean S.E.M (n ¼ 4).
short peptide derived from longer peptide exhibits higher receptor
affinity than the original peptides [36].
The above evidence suggested the combined cellular internalization promoting effect by the DGL polymer and leptin30 ligand.
The cellular uptake of DGLePEGeLeptin30/DNA NPs could be
inhibited by transferrin which was thought to be the typical marker
being internalized through clathrin-dependent pathway [37]. The
results were in good agreement with the previous study which
reported leptin receptor was internalized by clathrin-dependent
pathway [38,39] and confirmed leptin receptor-related endocytosis. Filipin acts as an inhibitor of caveolae mediated uptake by
binding cholesterol, an important component for caveolae formation [40]. The cellular internalization appears to be also mediated
through caveolae which is involved in the adsorption-mediated
endocytosis process [41]. Meanwhile, colchicine is used in this
study as macropinocytosis inhibitor [42]. The uptake of DGL/DNA
NPs was mediated by caveolae mediated endocytosis and macropinocytosis. The similar concepts were mentioned as macropinocytosis could also mediate the uptake of cationic carriers [43].
DGLePEGeLeptin30/DNA NPs endocytosis had relation to all the
three endocytosis pathway. It confirmed that the ligand and the
polymer both contribute to the cellular internalization of the NPs.
In addition, the confocal analysis of BCECs incubated with DGL/DNA
NPs indicated that EMA-labeled NPs could escape from endosomes
effectively at 1 h after incubation (data not shown).
DGLePEGeLeptin30/DNA NPs could be transported across in
vitro BBB model consisted of BCECs monolayers more effectively
than DGLePEG/DNA and DGL/DNA NPs as the permeability of
DGLePEGeLeptin30/DNA NPs was significantly higher than that of
DGLePEG/DNA NPs and DGL/DNA NPs from 10 min after incubation
(Fig. 6A). While transendothelial electrical resistance (TEER) and
permeability of 14C-sucrose (Fig. 6B) demonstrated that the integrity remained during the whole incubation process [35]. It could be
deduced the specific binding between leptin30 and leptin receptors
played an active role in the transcytosis of NPs.
In vitro transfection efficiency was evaluated in BV-2 microglia
cells. BV-2 cells as well as many other brain cells were reported to
expressed the long (OBRl) and short (OBRs) isoforms of the leptin
receptors [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 NPs
while equal to that of lipofectamine2000. It could be also explained
that leptin30 could bind leptin receptors on BV-2 cells and be
internalized by receptor mediated endocytosis. The DNA accumulation of DGLePEGeLeptin30/DNA NPs was found higher than that
of DGL/DNA NPs in brain after 80 min (Fig. 10). The report gene
expression at different regions in brain (cortical layer, hippocampus, caudate putamen and substantia nigra) has clearly
revealed the preferential localization of DGLePEGeLeptin30/DNA
NPs in the brain when compared with other two controls (Fig. 11).
The transfection efficiency in vivo was in accordance to the results
in BV-2 cells. A relative high report gene expression also showed
qualitatively in brain (Fig. 12A). The in vivo transfection ability
declined slightly compared with the in vitro results (Figs. 7 and 8).
The reasons may be the specific binding between targeting
DGLePEGeLeptin30/DNA NPs and leptin receptors on BBB was
interfered by endogenic leptin. It is reported the plasma level of
leptin in normal mice is about 280 nM [44] and doesn’t change
acutely with food administration [45], while the plasma level of
leptin30 modified NPs was estimated no more than 5000 nM in this
study. The targeting modification of NPs was sufficient enough to
exert their targeting effects. This result was in agreement with the
cellular uptake comparison result which demonstrated the uptake
efficiency altered slightly as the targeting modification increasing
from median to high. The accumulation in heart may be caused by
the property of DGL but not relate to the modification of Leptin30
since the report gene expressions were higher in DGL/DNA NPs
administration group than that in DGLePEG/DNA and
DGLePEGeLeptin30/DNA NPs administration groups (Fig. 12B). The
similar results were observed in gene delivery systems based on
PAMAM with different modification [12,13]. It is worth optimizing
the charge ratio N/P to achieve the best in vivo transfection efficiency further.
The cytotoxicity of the three series vector/DNA NPs at 2 h and
48 h in BCECs and BV-2 cells was relatively low at all the concentrations tested, while lipofectamine2000 didn’t show much cytotoxicity in this experiment (Fig. 9). Compared with some synthetic
polymers, DGL was thought to be less toxic and biodegradable [17],
hence possess better biocompatibility due to the peptide-like
linkage between the monomers. And the accumulative or longterm toxicity was relatively low. The above results further indicate
the potent brain-targeting ability of leptin30 modified PEGylated
DGL as the efficient gene vector.
5. Conclusions
DGLePEGeLeptin30 was synthesized and complexed with
plasmid DNA yielding nanoparticles. The mechanism of
DGLePEGeLeptin30/DNA NPs mediated brain-targeting is affected
by surface charge, PEGylation and leptin30 modification and could
be characterized as a clathrin and caveolae mediated energydepending endocytosis. Compared with in vitro transfection results
Y. Liu et al. / Biomaterials 31 (2010) 5246e5257
in vivo transfection ability may be interfered by endogenic leptin
slightly, the DGLePEGeLeptin30/DNA NPs were demonstrated to
be efficient enough for brain-targeting gene delivery. It could be
believed that DGLePEGeLeptin30 holds great promise as the nonviral vector for relatively safe and efficient brain-targeting gene
delivery.
Acknowledgements
[18]
[19]
[20]
[21]
The authors thank Pro. Jianhua Zhu (School of Pharmacy,
Fudan University) for the 125I-labeled work. This work was supported by the grant from National Basic Research Program
(2007CB935802) of China (973program), National Natural Foundation of China (30973652) and “Key new drug creation program”
2009ZX09310-006.
[22]
[23]
[24]
[25]
Appendix
Figures with essential colour discrimination. Certain figures in
this article, in particular Figs. 3,4,7,10 and 11 are difficult to interpret
in black and white. The full colour images can be found in the
online version, at doi:10.1016/j.biomaterials.2010.03.011.
References
[1] Perrion RJ, Fagan AM, Holtzman DM. Multimodal techniques for diagnosis and
prognosis of Alzheimer’s disease. Nature 2009;461:916e22.
[2] Rowe JB, Hughes L, Ghosh BC, Eckstein D, Williams-Gray CH, Fallon S, et al.
Parkinson’s disease and dopaminergic therapyddifferetial effects on movement, reward and cognition. Brain 2008;131:2094e105.
[3] Ranganath SH, Wang CH. Biodegradable microfiber implants delivering
paclitaxel for post-surgical chemotherapy against malignant glioma. Biomaterials 2008;29:2996e3003.
[4] Chen SH, Shine HD, Goodman JC, Grossman RG, Woo SL. Gene therapy for
brain tumors: regression of experimental gliomas by adenovirus-mediated
gene transfer in vivo. Proc Natl Acad Sci U S A 1994;91:3054e7.
[5] Luten J, van Nostrum CF, De Smedt SC, Hennink WE. Biodegradable polymers as
non-viral carriers for plasmid DNA delivery. J Control Release 2008;126:97e110.
[6] Mochizuki H, Yasuda T, Mouradian MM. Advances in gene therapy for
movement disorders. Neurotherapeutics 2008;5:260e9.
[7] Lesniak MS. Gene therapy for malignant glioma. Expert Rev Neurother
2006;6:479e88.
[8] Lehrman S. Virus treatment questioned after gene therapy death. Nature
1999;401:517e8.
[9] Choi S, Lee KD. Enhanced gene delivery using disulfide-crosslinked low
molecular weight polyethylenimine with listeriolysin o-polyethylenimine
disulfide conjugate. J Control Release 2008;131:70e6.
[10] Diez S, Tros de llarduya C. Versatility of biodegradable poly(D,L-lactic-co-glycolic acid) micropheres for plasmid DNA delivery. Eur J Pharm Biopharm
2006;63:188e97.
[11] Lee JH, Lim YB, Choi JS, Lee Y, Kim TI, Yoon JK, et al. Polyplexes assembled with
internally quaternized PAMAM-OH dendrimer and plasmid DNA have a neutral
surface and gene delivery potency. Bioconjug Chem 2003;14:1214e21.
[12] Huang RQ, Ke WL, Liu Y, Jiang C, Pei YY. The use of lactoferrin as a ligand for
targeting the polyamidoamine-based gene delivery system to the brain.
Biomaterials 2008;29:238e46.
[13] Liu Y, Huang RQ, Ke WL, Jiang C. Brain-targeting gene delivery and cellular
internalization mechanisms for modified rabies virus glycoprotein RVG29
nanoparticles. Biomaterials 2009;30:4195e202.
[14] Ke WL, Shao K, Huang RQ, Liu Y, Li JF, Jiang C. Gene delivery targeted to the
brain using an angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer. Biomaterials 2009;30:6976e85.
[15] Klok HA, Juan RH. Dendritic-graft polypeptides. Macromolecules
2002;35:8718e23.
[16] Cottet H, Martin M, Papillaud A, Souaid E, Collet H, Commeyras A. Determination of dendrigraft poly-L-lysine diffusion coefficients by taylor dispersion
analysis. Biomacromolecules 2007;8:3235e43.
[17] Tsogas I, Theodossiou T, Sideratou Z, Paleos CM, Collet H, Rossi JC, et al.
Interaction and transport of poly(L-lysine) dendrigrafts through liposomal and
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
5257
cellular membranes: the role of generation and surface functionalization.
Biomacromolecules 2007;8:3263e70.
Seong LL, Shu W. An endosomolytic tat peptide produced by incorporation of
histidine and cysteine residues as a nonviral vector for DNA transfection.
Biomaterials 2008;29:2408e14.
Vykhodtseva N, McDannold N, Hynynen K. Progress and problems in the
application of focused ultrasound for blood-brain barrier disruption. Ultrasonics 2008;48:279e96.
Patel MM, Goyal BR, Bhadada SV, Bhatt JS, Amin AF. Getting into the brain:
approaches to enhance brain drug delivery. CNS Drugs 2009;1:35e8.
Zorko M, Langel U. Cell-penetrating peptides: mechanism and kinetics of
cargo delivery. Adv Drug Deliv Rev 2005;57:529e45.
Chang J, Jallouli Y, Kroubi M, Yuan XB, Feng W, Kang CS, et al. Characterization
of endocytosis of transferrin-coated PLGA nanoparticles by the bloodebrain
barrier. Int J Pharm 2009;379:285e92.
Tritos NA, Mantzoros CS. Leptin: its role in obesity and beyond. Diabetologia
1997;40:1371e9.
Houseknecht KL, Baile CA, Matteri RL, Spurlock ME. The biology of leptin:
a review. J Anim Sci 1998;76:1405e20.
Golden PL, Maccagnan TJ, Pardridge WM. Human bloodebrain barrier leptin
receptor. J Clin Invest 1997;99:14e8.
Tartaglia LA. The leptin receptor. J Biol Chem 1997;272:6093e6096.
Barrett GL, Trieu J, Naim T. The identification of leptin-derived peptides that
are taken up by the brain. Regul Pept 2009;155:55e61.
Banks WA, Kastin AJ, Huang W, Jaspan JB, Maness LM. Leptin enters the brain
by a saturable system independent of insulin. Peptides 1996;17:305e11.
Tang CH, Lu DY, Yang RS, Tsai HY, Kao MC, Fu WM. Leptin-induced IL-6
production is mediated by leptin receptor, insulin receptor substrate-1,
phosphatidylinositol 3-kinase, Akt, NF-kB, and p300 pathway in microglia.
J Immunol 2007;179:1292e302.
Xie Y, Ye LY, Zhang XB, Cui W, Lou JN, Nagai T. Transport of nerve growth
factor encapsulated into liposomes across the blood-brain barrier: in vitro and
in vivo studies. J Control Release 2005;105:106e19.
Huang RQ, Qu YH, Ke WL, Zhu JH, Pei YY, Jiang C. Efficient gene delivery
targeted to the brain using a transferrin-conjugated polyethyleneglycolmodified polyamidoamine dendrimer. FASEB J 2007;21:1117e25.
Xie Y, Ye LY, Zhang XB, Hou XP, Lou JN. Establishment of an in vitro model of
braineblood barrier. Beijing Da Xue Xue Bao 2004;36:435e8.
Irvine JD, Takahashi L, Lockhart K, Cheong J, Tolan JW, Selick HE. MDCK
(Madin-Darby canine kidney) cells: a tool for membrane permeability
screening. J Pharm Sci 1999;88:28e33.
Pan J, Feng SS. Targeted delivery of paclitaxel using folate-decorated poly
(lactide)-vitamin E TPGS nanoparticles. Biomaterials 2008;29:2663e72.
Pardridge WM, Triguero D, Yang J, Cancilla PA. Comparison of in vitro and in
vivo models of drug transcytosis through the bloodebrain barrier. J Pharmacol
Exp Ther 1990;253:884e91.
Roumy M, Gouarderes C, Mazarguil H, Zajac JM. Are neuropeptides FF and SF
neurotransmitters in the rat? Biochem Biophys Res Commun
2000;275:821e4.
Huth US, Schubert R, Peschka-Suss R. Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bioimaging. J Control Release 2006;110:490e504.
Barr VA, Lane K, Taylor SI. Subcellular localization and internalization of the
four human leptin receptor isoforms. J Biol Chem 1999;274:21416e24.
Belouzard S, Rouille Y. Ubiquitylation of leptin receptor OB-Ra regulates its
clathrin-mediated endocytosis. EMBO J 2006;25:932e42.
Sharma DK, Choudhury A, Singh RD, Wheatley CL, Marks DL, Pagano RE.
Glycosphingolipids internalized via caveolar-related endocytosis rapidly
merge with the clathrin pathway in early endosomes and form microdomains
for recycling. J Biol Chem 2003;278:7564e72.
Thole M, Nobmanna S, Huwyler J, Bartmann A, Fricker G. Uptake of
cationzied albumin coupled liposomes by cultured porcine brain microvessel endothelial cells and intact brain capillaries. J Drug Target
2002;10:337e44.
Liu J, Shapiro JI. Endocytosis and signal transduction: basic science update.
Biol Res Nurs 2003;5:117e28.
Morille M, Passirani C, Vonabourg A, Clavreul A, Benoit JP. Progress in
developing cationic vectors for non-viral systemic gene therapy against
cancer. Biomaterials 2008;29:3477e96.
Song YM, Chen MD. Effect of melatonin administration on plasma leptin
concentration and adipose tissue leptin secretion in mice. Acta Biol Hung
2009;60:399e407.
Korbonits M, Trainer PJ, Little JA, Edwards R, Kopelman PG, Besser GM. Leptin
levels do not change acutely with food administration in normal or obese
subjects, but are negatively correlated with pituitary-adrenal activity. Clin
Endocrinol 1997;46:751e7.