Identification of Peptide Ligands for Targeting to the Blood-Brain Barrier
ABSTRACT Transport of drugs to the brain is limited by the blood-brain barrier. New, specific brain endothelium ligands can facilitate brain-specific delivery of drugs.
We used phage display in an in situ brain perfusion model to screen for new brain endothelium peptide ligands.
Two phage clones, displaying 15 amino acid-peptides (GLA and GYR) that were selected for brain binding in the mouse model, showed significant binding to human brain endothelium (hCMEC/D3), compared to a random control phage. This binding was not seen for other human endothelial cells (HUVEC). Binding to hCMEC/D3 cells was dose dependent. When phage GLA and GYR were individually perfused through the murine brain, their ability to bind to the brain was 6-fold (GLA) and 5-fold (GYR) higher than the control phage. When compared to lung perfusion, phage showed an 8.5-fold (GYR) and 48-fold (GLA) preference for brain over lung compared to the control.
These results indicate that two new peptide ligands have been identified that may be used for specific targeting of drugs to the blood-brain barrier.
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ABSTRACT: A variety of therapeutic proteins have shown potential to treat central nervous system (CNS) disorders. Challenge to deliver these protein molecules to the brain is well known. Proteins administered through parenteral routes are often excluded from the brain because of their poor bioavailability and the existence of the blood-brain barrier (BBB). Barriers also exist to proteins administered through non-parenteral routes that bypass the BBB. Several strategies have shown promise in delivering proteins to the brain. This review, first, describes the physiology and pathology of the BBB that underscore the rationale and needs of each strategy to be applied. Second, major classes of protein therapeutics along with some key factors that affect their delivery outcomes are presented. Third, different routes of protein administration (parenteral, central intracerebroventricular and intraparenchymal, intranasal and intrathecal) are discussed along with key barriers to CNS delivery associated with each route. Finally, current delivery strategies involving chemical modification of proteins and use of particle-based carriers are overviewed using examples from literature and our own work. Whereas most of these studies are in the early stage, some provide proof of mechanism of increased protein delivery to the brain in relevant models of CNS diseases, while in few cases proof of concept had been attained in clinical studies. This review will be useful to broad audience of students, academicians and industry professionals who consider critical issues of protein delivery to the brain and aim developing and studying effective brain delivery systems for protein therapeutics.Journal of Controlled Release 06/2014; · 7.26 Impact Factor
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ABSTRACT: Personalized medicine is at the forefront of cancer diagnosis and therapy. Molecularly targeted therapies such as trastuzumab and tamoxifen have enhanced prognosis of patients with cancers expressing ERBB2 and the estrogen receptor, respectively. One obstacle to targeted therapy is the development of resistance. A targeted peptide that could distinguish resistance-susceptible cancer would aid in treatment. BT-474 human breast cancer cells can be resistant to both tamoxifen and trastuzumab, and may serve as a model for malignancies in which targeted therapy may not work. Bacteriophage (phage) display is a combinatorial technology that has been used to isolate peptides that target a specific cancer subtype. It was hypothesized that in vivo phage display could be used to select a peptide for SPECT imaging of BT-474 human breast cancer xenografts. A phage library displaying random 15 amino acid peptides was subjected to four rounds of selection, after which 14 clones were analyzed for BT-474 binding and specificity. One phage clone, 51, demonstrated superior binding and specificity, and the displayed peptide was synthesized for in vitro characterization. Peptide 51 bound specifically to BT-474 cells with an EC50 = 2.33 µM and was synthesized as a DOTA-conjugated peptide and radiolabeled with (111)In for in vitro and in vivo analysis. The radiolabeled peptide exhibited an IC50 = 16.1 nM to BT-474 cells and its biodistribution and SPECT imaging in BT-474 xenografted mice was analyzed. Although tumor uptake was moderate at 0.11% ID/g, SPECT imaging revealed a distinct tumor vasculature binding pattern. It was discovered that peptide 51 had an identical 5 amino acid N-terminal sequence to a peptide, V1, which bound to Nrp1, a tumor vasculature protein. Peptide 51 and V1 were examined for binding to target cells, and 51 bound both target and endothelial cells, while V1 only bound endothelial cells. Truncated versions of 51 did not bind BT-474 cells, demonstrating that the targeting ability of 51 was independent of the homologous V1 sequence. These results demonstrate that in vivo phage display can effectively identify a peptide that specifically targets a breast cancer cell line that is susceptible to targeted therapy resistance.American Journal of Nuclear Medicine and Molecular Imaging 01/2014; 4(5):435-47. · 3.25 Impact Factor
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ABSTRACT: A series of tri[(p-carboranylmethylthio)tetrafluorophenyl]porphyrin conjugates of linear and branched polyamines, glucose, arginine, tri(ethylene glycol), and peptide Tyr-D-Arg-Phe-β-Ala (TAPA) were synthesized. These conjugates were investigated for their BBB permeability in human hCMEC/D3 brain endothelial cells as model, and their cytotoxicity and uptake in human glioma T98G cells. For comparison purposes, a symmetric tetra[(p-carboranylmethylthio)tetrafluorophenyl]porphyrin was also synthesized and its crystal structure was obtained. All porphyrin conjugates show low dark cytotoxicity (IC50 > 400 μM) and low phototoxicity (IC50>100 μM at 1.5 J/cm2) toward T98G cells. All conjugates were efficiently taken up by T98G cells, particularly the cationic polyamine and arginine conjugates, and localized in multiple cell organelles, including the mitochondria and lysosomes. All compounds showed relatively low in vitro BBB permeability compared with lucifer yellow, probably due to their higher molecular weight, hydrophobicity and tendency for aggregation in solutions. Among this series, the branched polyamine and TAPA conjugates showed the highest permeability coefficients, while the glucose conjugate showed the least.Journal of medicinal chemistry. 07/2014;
Identification of Peptide Ligands for Targeting to the Blood-Brain Barrier
Inge van Rooy,1,6Serpil Cakir-Tascioglu,1Pierre-Olivier Couraud,2,3Ignacio A. Romero,4Babette Weksler,5
Gert Storm,1Wim E. Hennink,1Raymond M. Schiffelers,1and Enrico Mastrobattista1
Received October 14, 2009; accepted January 5, 2010; published online February 17, 2010
Purpose. Transport of drugs to the brain is limited by the blood-brain barrier. New, specific brain
endothelium ligands can facilitate brain-specific delivery of drugs.
Methods. We used phage display in an in situ brain perfusion model to screen for new brain endothelium
Results. Two phage clones, displaying 15 amino acid-peptides (GLA and GYR) that were selected for
brain binding in the mouse model, showed significant binding to human brain endothelium (hCMEC/
D3), compared to a random control phage. This binding was not seen for other human endothelial cells
(HUVEC). Binding to hCMEC/D3 cells was dose dependent. When phage GLA and GYR were
individually perfused through the murine brain, their ability to bind to the brain was 6-fold (GLA) and 5-
fold (GYR) higher than the control phage. When compared to lung perfusion, phage showed an 8.5-fold
(GYR) and 48-fold (GLA) preference for brain over lung compared to the control.
Conclusions. These results indicate that two new peptide ligands have been identified that may be used
for specific targeting of drugs to the blood-brain barrier.
KEY WORDS: blood-brain barrier; brain targeting; peptide ligand; phage display; targeting ligand.
Transport of drugs to the brain is limited by the presence
of the blood-brain barrier (BBB). This barrier is formed by
specialized endothelial cells and supported by other cell
types, such as astrocytes and pericytes (1). The BBB regulates
homeostasis of the brain and is selectively permeable for the
uptake and efflux of ions, nutrients and metabolites (2). Drug
transport via the paracellular route is prevented by tight
junctions between the endothelial cells. Free diffusion via the
transcellular route is accessible only to lipophilic compounds
smaller than approximately 400 Da (3). This limits the
treatment of many brain diseases, such as Alzheimer’s disease
and Parkinson’s disease. Even in certain pathological situa-
tions where the BBB is partly disrupted, drug transport
remains limited (4).
To overcome this limitation, carrier systems, such as
nanoparticles, have been used to deliver drugs to the brain.
Antibody ligands have been successfully coupled to the
nanoparticles to target the brain endothelial cell receptors
(5,6). The main receptors that have been targeted are the
transferrin receptor (7) and the insulin receptor (8). However,
these receptors are not brain specific. They are widely
expressed in peripheral organs (9), limiting their selectivity
and applicability as brain-targeting receptors. Therefore,
identification of new brain targets is needed to deliver drugs
more selectively into the brain. The objective of this study
was to identify new targeting ligands that can be used for
active targeting of drug delivery systems to the brain, aiming
for selectivity for brain endothelium.
Peptides are highly suitable for ligand screening, when
expressed on a phage display system, and they have been
widely used as targeting ligands (10). Phage display is a
powerful technology for ligand identification (11–14). Large
libraries expressing peptides or proteins can be screened for
target affinity (15). We used a random 15-mer peptide library
to identify brain-targeting peptide ligands. In vivo, blood-
brain barrier endothelial cells get stimulated by their
surrounding cells (16,17) and intraluminal blood flow (18).
This contributes to the complexity of the blood-brain barrier
and regulates the expression of specific receptors at the cell
surface in a polarized fashion. For this reason, ligand screen-
ing should preferably be performed in vivo. However, as in
vivo phage screening in humans was impossible for ethical
reasons, we chose to pre-select phage in a mouse perfusion
1Department of Pharmaceutics, Utrecht Institute for Pharmaceutical
Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB,
Utrecht, The Netherlands.
2Institut Cochin, Université Paris Descartes, CNRS (UMR 8104),
3Inserm, U567, Paris, France.
4Department of Biological Sciences, The Open University, Milton
5Department of Medicine, Weill Medical College, New York, New
6To whom correspondence should be addressed. (e-mail: i.vanrooy@
Pharmaceutical Research, Vol. 27, No. 4, April 2010 (#2010)
0724-8741/10/0400-0673/0#2010 The Author(s). This article is published with open access at Springerlink.com
model to enrich for peptides recognizing brain endothelial
cells in their natural environment. Because the brain remains
intact, the endothelial cells keep their polarity and their
contact with the surrounding cells, maintaining expression of
relevant receptors. After the pre-selection for in vivo relevant
receptors, identified ligands were tested for cross-reactivity
with human cell-surface receptors expressed by human
endothelial cells in vitro. In this way, the complexity of the
in vivo BBB and the significance of the human BBB were
MATERIALS AND METHODS
Phage Library and Bacteria
Filamentous phage fd-tet, which confers tetracycline
resistance on the host (19), was used for this study. Random
peptide library f3-15mer (20), containing 2.5×108primary
clones (GenBank Accession AF246445) with foreign 15-mer
peptide displayed on pIII was provided by George P. Smith
(Columbia, MO). Kanamycin resistant E. coli K91BluKan
(K91BK) host bacteria were also provided by George P.
Smith. The K91BK sex is Hfr Cavalli, resulting in deployment
of the F pilus, the attachment site for filamentous phage
In situ phage display screening was performed in male,
6–8 week-old C57Bl/6 mice (Charles River, The Netherlands).
Perfusion of single clones was done in male 28–32 g Balb/c mice
(Harlan, Horst, The Netherlands). Food and water were
supplied ad libitum. Animal studies were performed according
to national regulations and were approved by the local animal
experiments ethical committee.
In Situ Brain Perfusion of Phage
Forty units of heparin (Sigma) were injected i.v. into the
tail vein to prevent coagulation of blood inside the micro-
vessels (21). Five minutes after injection, mice were sacrificed
by CO2asphyxiation. Next, thorax was opened, and bulldog
clamps with 8×1.2 mm serrated jaws (WPI, Berlin, Germany)
were placed on descending aorta (left from the carotid arteries)
and pulmonary veins and arteries. Inferior caval vein was cut
to allow outflow of perfused fluids. A 26G needle, connected
to a peristaltic pump, was inserted into the left ventricle of the
heart (22). Mice were initially perfused with 1.5 ml Hanks’
Balanced Salt Solution (HBSS, pH 7.0–7.4, Invitrogen,
Carlsbad, CA) in all experiments. Directly afterwards, mice
were perfused with phage. Upon the three selection rounds,
1 ml of phage library in HBSS (8.9×1011TU, 3.1×1011TU,
and 4.8×1010TU for the first, second and third selection
rounds, respectively) were perfused (input). To wash away
non-binders, 3.5 ml HBSS was perfused directly afterwards.
For the third selection round, HBSS + 1% FBS was used
instead of HBSS. For perfusions of selected phage (single
clones), 750 µl of phage (∼1010TU) in HBSS + 1% FBS was
perfused, directly followed by 2 ml of HBSS + 1% serum to
wash. Peristaltic pump speed was 200 μl/min. Directly after
perfusion, brains were taken out. For the phage display
screening, phage were isolated from the brain (see below).
Phage Isolation from Brain
After phage display selection rounds, brain cerebrum
was ground in 600 μl 100 mM triethanolamine (Fluka,
Munich, Germany) and centrifuged for 10 min at 3300 ×g.
Supernatant was transferred to a new tube, neutralized with
300 μl 1 M Tris pH 7.4, and centrifuged for 5 min at 3300 ×g.
Late log phase K91BK were obtained by inoculation of 10 ml
of terrific broth with 100 μl of an overnight culture, and
incubation for 4 h in a shaker incubator (250 rpm, 37°C).
When late log phase was reached (when the OD600of a 10×
dilution reached 0.2 on a spectrophotometer), shaking was
slowed down to 75 rpm for 5 min to allow sheared F pili to
regenerate. Brain supernatant was incubated with late log
phase K91BK for 10 min to allow phage to infect the bacteria.
The infected bacteria were added to 40 ml LB Broth (Sigma)
containing 0.2 μg/ml tetracycline and amplified for 30 min in a
shaker incubator (250 rpm, 37°C). Two hundred μl of the
infected K91BK were spread on LB agar plates for titration.
Tetracycline concentration was then brought to 20 μg/ml, and
phage were amplified overnight in the shaker incubator.
Cultures were cleared of bacteria by two 10 min centrifuga-
tions at 3000 and 8000 ×g. To the cleared culture 0.15 volume
PEG/NaCl (16.7% /3.3 M stock) was added (PEG 8000,
Promega, Breda, The Netherlands). Phage were precipitated
overnight at 4°C. Precipitated phage were collected by
centrifugation for 15 min at 17500 ×g at 4°C. Pellets were
dissolved in 1 ml TBS and precipitated again with 0.15 vol
PEG/NaCl for at least 1 h at 4°C. Phage were collected by
centrifugation for 5 min at 16000 ×g. Pellets were dissolved
in 400 μl TBS and stored at 4°C. Two hundred μl of
purified phage + 800 μl HBSS were used for the next
selection round. Three successive selection rounds were
done in total. After the third selection round, brain
cerebrum was separated into anterior and posterior part.
Phage were separately isolated from both parts. Titration
of input phage was performed as described below. After
perfusion of single phage clones, whole brains were ground
in 1,000 μl triethanolamine and centrifuged for 10 min at
3300 ×g. Two hundred μl supernatant was transferred to a
new tube, and neutralized with 100 μl 1 M Tris pH 7.4.
Titration of both input and output phage was performed as
Titration of Phage
Transducing units (TU) were determined by titration.
Phage were serially diluted in 0.1% (w/v) gelatin in TBS. Ten
μl of phage dilution were incubated with 10 μl of late log
phase K91BK for 10 min at room temperature. One ml LB,
containing 0.2 μg/ml tetracycline, was added. This was
incubated for 40 min in a shaker incubator (250 rpm, 37°C).
Two-hundred μl of the infected K91BK were spread on LB
agar plates containing 40 μg/ml tetracycline and 100 μg/ml
kanamycin. Plates were incubated overnight at 37°C. The
next day, the number of colonies was counted, and the number
of transducing units was calculated. Input phage were titrated
as well at the same time for recovery percentage calculations.
674van Rooy et al.
Immunohistochemistry Brain Cryosections
Frozen untreated brains were coated with OCT embed-
ding matrix (Cellpath, Newtown, UK). Sections were cut
along the sagittal plane using low-profile microtome blades
(Leica, Heidelberg, Germany) on a CM3050 cryostat (Leica).
Sections were transferred onto Superfrost Plus glass slides
(Menzel, Germany). Section thickness was 5 μm. They were
dried overnight at room temp and stored at −80°C. Upon use,
sections were allowed to thaw for 20 min, fixed in acetone for
10 min, and air dried for 30 min. Separate sections were en-
circled with a liquid-repellent marker. All incubations were
done in a moist environment, and incubation volumes were
90 μl/section. Non-specific binding sites and endogenous biotin
were blocked simultaneously with avidin/biotin-blocking solu-
tion in PBS + 5% FCS (Vector Laboratories, Inc., Burlingame,
USA), according to manufacturer’s instructions. Brain sections
were incubated with either the enriched library or the unse-
lected library (both 2.37×1013V/ml) for 1 h at room
temperature. Sections were incubated with 0.8 μg/ml
biotinylated anti-phage antibody (Abcam, Cambridge, UK) for
1 h. Thereafter, sections were incubated with 5 μg/ml alexa568-
conjugated streptavidin (Invitrogen) for 35 min. Sections were
mounted in FluorSave (Calbiochem, San Diego, CA) and
viewed under a Nikon Eclipse TE2000-U epi-fluorescence
microscope (Nikon, Tokyo, Japan) at a 20× magnification.
Peptide sequences were aligned to check for peptides’
consensus sequences along the 15 amino acids. Software used
was Vector NTI Advance 10 AlignX (Invitrogen).
Human brain endothelial cells (hCMEC/D3) were
obtained from Institut National de la Santé et de la
Recherche Médicale (INSERM, Paris, France). Cells were
cultured at 37°C, 5% CO2, in EBM-2 basal medium
supplemented with EGM-2 MV BulletKit (Lonza, Basel,
Switzerland), containing growth factors and 2.5% FBS.
Additionally, 10 mM HEPES, 1 ng/ml bFGF (Invitrogen),
and antibiotics (Penicillin and Streptomycin) were added.
hCMEC/D3 cells were grown on surfaces coated with 100 μg/
ml Rat tail collagen type 1 (BD Biosciences). Prior to use, cell
culture medium was replaced by cell differentiation medium,
consisting of EBM-2 basal medium supplemented with 2.5%
FBS, 1.4 μM hydrocortisone, 1 ng/ml bFGF, 10 mM HEPES,
and antibiotics. Cells were grown to a monolayer in differ-
entiation medium for one week. Medium was replaced every
Human umbilical vein endothelial cells (HUVEC) were
cultured at 37°C, 5% CO2, in EBM-2 basal medium
supplemented with antibiotics and EGM-2 BulletKit (Lonza),
containing growth factors and 2% FBS.
In Vitro Binding of Single Phage Clones
Cells were seeded at 3.4·105cells/well in 12-well plates.
Single phage clones were diluted to ∼1010TU/ml in 25 mM
HEPES in HBSS, and 1 ml phage solution was incubated with
either hCMEC/D3 or HUVEC cells for 1 h at 37°C. The
buffer containing unbound phage was removed, and cells
were washed three times with PBS. Cells were washed three
times with 1 ml of 0.2 M glycine (pH 2.2) to elute surface-
bound phage (23,24). Three ml wash eluate was collected and
neutralized with 0.45 ml 1 M tris (pH 9.1). Cells were washed
again with 1 ml of PBS and were subsequently lysed with
0.25 ml 100 mM triethanolamine for 10 min (25,26). Cell
lysates were collected and neutralized with 0.0625 ml 1 M tris
(pH 7.4). The neutralized wash eluates (binding fraction) and
cell lysates (strong binding/uptake fraction) were titrated as
described above. Input phage were titrated as well at the
same time for recovery percentage calculations.
Immunohistochemistry hCMEC/D3 Cells
hCMEC/D3 cells were grown to a monolayer on rat tail
collagen coated Lab-Tek chamber slides (Nunc, Rochester,
NY). Single phage clones were diluted to 5·1012V/ml in
HBSS, and 100 μl phage solution or 100 μl HBSS only was
incubated with the cells for 1 h at 37°C. The buffer containing
unbound phage was removed, and cells were washed three
times with PBS. Cells were fixed in 4% formaldehyde for
30 min. Cells were washed two times with PBS and incubated
with 0.8 μg/ml biotinylated anti-phage antibody for 1 h.
Thereafter, cells were incubated with 5 μg/ml alexa568-
conjugated streptavidin for 35 min, and washed three
times. Cells were incubated with 300 nM DAPI (Invitrogen)
for 2 min and washed three times with demi water. Cells
were mounted in FluorSave and viewed under a Nikon
Eclipse TE2000-U epi-fluorescence microscope at a 20×
In Situ Lung Perfusion of Phage
In situ lung perfusion was performed in the same way as
was done for brain perfusions of single clones, with the only
modification being the placement of the bulldog clamps. The
ascending aorta was clamped, and phage were perfused
through the left ventricle of the heart. Inferior caval vein
was cut to allow outflow of perfused fluids. Phage isolation
and titration were also performed in the same way as was
done for the brain perfusions.
Data were analyzed using Graphpad Prism 4 for
Windows software (Graphpad Software, San Diego, CA).
In situ phage display screening was used as a tool to
search for new blood-brain-barrier-binding peptides. Screen-
ing was performed by perfusion of a 15 amino acid random
peptide phage display library through the murine brain in
situ. Phage perfusion (27,28) was chosen over intravenous
injection, because clearance of phage occurs rapidly via the
major organs of the reticuloendothelial system (mainly liver
and spleen), resulting in a half-life of fd-tet of only 12 min
(29). Therefore, phage were directly perfused via the heart
through the brain, preventing loss to other organs, and
675Peptide Ligands for BBB Targeting
ensuring a high fraction of individual peptide-displaying
phage to reach the target tissue.
Various in situ brain perfusion methods have been
developed (30). Most models have been established in rats,
but murine models have been used as well (22,31,32). Most
perfusion models have been used to study brain uptake of
substrates and were performed under anesthesia. Since
filamentous phage are approximately 1 μm in size (33), they
are not likely to be taken up into the brain, and the perfusion
allows screening for binding rather than for internalizing
peptides. Therefore, body temperature maintenance was not
required, and perfusion was performed at room temperature,
postmortem. The large majority of brain proteins are stable
during a postmortem interval of up to 4 h at 25°C (34). As
perfusions were completed within 30 min after sacrifice, the
postmortem state was not expected to cause significant
changes in brain microvessel morphology.
Phage were infused via the heart. The major advantage
compared to direct infusion into the brain is that the phage
have to travel some distance before they reach the brain. On
their way, they already encounter endothelial cells: the
endothelium of the aorta and carotid arteries. Phage with
affinity to ubiquitous endothelial receptors are given the
opportunity to bind to these cells and will not reach the brain.
This negative selection filters out unspecific binders and
allows for the identification of peptides that bind more
specifically to brain endothelium (35,36).
Phage display screening was performed by perfusion of a
mouse brain with 8.9×1011Transducing Units (TU) in Hanks’
Balanced Salt Solution (HBSS), containing 1 g/L glucose, to
keep the brain in a viable state. Phage were perfused for
5 min. Phage incubation times of 5–15 min are common and
have been successful in vivo (11,37–40). Non-binders were
washed away by perfusion of HBSS. The brain was taken out,
and phage were isolated from the cerebrum and submitted to
the next selection round. During the third selection round,
1% FBS was added to the HBSS to limit unspecific
interactions. Because the infused fluid was not evenly
perfused throughout the brain (see supporting information
paragraph), cerebrum was divided into its anterior (weakly
perfused) and posterior (strongly perfused) side after the
third selection round to investigate whether different ligands
could be identified in the different brain parts.
After three successive selection rounds, 17 clones
isolated from the anterior part and 17 clones isolated from
the posterior part of the brain were sequenced (Table I). Two
of the sequences were found twice, once in the anterior part
and once in the posterior part. However, most of the
sequences were found only once. There was no clear
discrimination in amino acid sequence between clones
isolated from the different brain parts. Alignment analysis
revealed no consensus sequences. This is not surprising,
considering the complexity of the brain endothelium surface
and the many possible targets it may contain.
After selection, the enriched library was tested for its
ability to bind to the brain by immunohistochemistry.
Frozen untreated murine brains were sectioned, and both
the unselected (naive) and enriched library were incubated
on the brain sections. Bound phage were stained with a
labeled anti-phage antibody. Fluorescence microscopy
revealed that the enriched library showed enhanced bind-
ing to the brain sections, compared to the naive library
(Fig. 1). The staining was comparable in the anterior and
The peptides were selected for affinity to the in vivo
relevant murine brain. However, for clinical applications,
affinity for human endothelium is desired. Cross-reactivity
between mouse and human has been shown for phage display
selected peptides (39). Therefore, a number of selected and
sequenced phage clones were tested for their ability to bind
human brain endothelial cells (picked at random from
Table IA, B). Additionally, three clones were randomly
chosen from the naive library as a negative control
(Table IC). The hCMEC/D3 cell line was used to represent
the human blood-brain barrier. hCMEC/D3 is a stable, fully
characterized, well-differentiated human brain endothelial
cell line (41,42). Phage were added to the cells and
incubated for 1 h at 37°C (23,25,43). Cell-associated phage
were recovered in two steps (25,26)—first, by washing the
cell surface with a low pH glycine buffer (stripping surface-
bound phage) and subsequently by lysing the cells
(collecting strongly bound and internalized phage). The
percentages of recovered phage (percentage from input)
are shown in Fig. 2. Fig. 2A shows that only seven out of
nineteen selected clones showed higher binding to the cells
compared to the highest random control clone (SVE). This
may be due to non-specific electrostatic interactions of the
peptides with the cell membranes, limiting discrimination
between specific and non-specific binders. However, when
looking at strong binding/internalization, these non-specific
interactions are at background level. As shown in Fig. 2B,
seventeen out of nineteen selected clones showed a higher
cell association as compared to the highest random control
clone (YLR), indicating that peptides were identified that
can specifically associate with brain endothelial cells. When
the anterior and posterior sides of the cerebrum are
compared, phage isolated from the posterior side showed
significantly higher cell association (Fig. 2B) than phage
isolated from the anterior side. This can be correlated to the
perfusion pattern seen upon Evans blue perfusion, which
showed better perfusion through the posterior side of the
brain (see supporting information paragraph). Likely, more
individual phage reached the posterior side, increasing the
selection strength and resulting in stronger binding phage.
Of all clones tested in vitro, phage GLA and GYR showed
the best brain endothelium binding. Therefore, GLA and
GYR were chosen for further testing.
In order to investigate dose dependency, binding of
phage GLA, GYR, and negative control YLR to the
hCMEC/D3 cells was determined for different concentrations
of input phage. Fig. 3 shows that the phage bound to the cells
in a dose-dependent manner. Significantly more GLA and
GYR were bound to the cells compared to control phage
The results indicate that phage GLA and GYR show
affinity for the hCMEC/D3 brain endothelium. We tested in
vitro whether this affinity was brain-specific or whether these
phage have affinity for other endothelial cells as well.
Therefore, phage association was tested on non-brain
endothelium: human umbilical vein endothelial cells
(HUVEC). Best hCMEC/D3 binding phage GLA and
GYR were tested, as well as poor hCMEC/D3 binding
676 van Rooy et al.
Fig. 1. Naive library (A) and selected (B) phage incubated on murine brain cryosections. A view on the
posterior side of the cerebrum is shown. Phage were labeled with alexa-568. Magnification 20×.
Table I. Sequenced Phage Clones. Clones After 3 Selection Rounds, Isolated from Anterior (A) and Posterior (B) Cerebrum. Control Clones
(C), Randomly Taken from Naive Library. Isoelectric Point (pI) and Charge at pH 7 Were Calculated by Vector NTI Advance 10 AlignX
Software. Clones Tested In Vitro (y = yes) are Shown in Fig. 2
CloneNameSequenceFrequencypI charge at pH 7Tested in vitro
A. Clones isolated from anterior cerebrum
B. Clones isolated from posterior cerebrum
C. Random control clones
677Peptide Ligands for BBB Targeting
phage GTW, and random control phage YLR. Phage GLA
and GYR show significantly less affinity to HUVECs
compared to hCMECs (Fig. 4), indicating that these phage
do not have affinity to all endothelial cells and seem to be
To confirm their ability to bind to brain endothelium in
situ, GLA, GYR, and random control phage RVR were
individually perfused through the brain again in the in situ
perfusion model. After perfusion and washing, phage were
recovered from the brain, and the percentages of the input
were determined (Fig. 5). Phage GYR showed a 5.0 times
higher affinity to the brain than random control phage RVR.
Phage GLA showed a 5.9 times higher affinity. This confirms
that brain-binding phage were identified. Although the
recovery percentages seem low, they are comparable to in
vivo phage recovery percentages found by others (39).
After establishment of brain specificity in vitro, specific-
ity was investigated in situ as well. The lung was considered a
suitable control perfusion organ. Just like brain, lung
comprises microvascular endothelium, and affinity of the
phage for this endothelium was tested. Phage GLA, GYR,
and control phage RVR were perfused through murine lungs
and were processed in exactly the same way as was done for
the brain perfusion. As is shown in Fig. 6, GLA binds
significantly less to the lungs compared to control phage
RVR. Binding of GYR is comparable to RVR. The negative
charge of GLA is likely to be the cause of lower affinity to the
From both brain and lung recovery percentages, the
preference ratio for brain over lung compared to RVR was
calculated. Phage GYR showed a 8.5-fold preference, and
phage GLA showed a 48-fold preference for brain over lung
compared to the control, implying specific affinity of these
phage-displayed peptides to brain endothelium.
To visualize the binding of phage GLA and GYR to
human brain endothelial cells, hCMEC/D3 cells were incu-
bated with single phage clones for 1 h at 37°C. After washing,
bound phage were stained with a labeled anti-phage antibody.
Fluorescence microscopy showed phage staining throughout
the cell monolayer. The pictures clearly show enhanced
binding of selected phage GLA and GYR compared to the
control phage RVR (Fig. 7). This is in agreement with the
brain-barrier-targeting properties of these selected phage-
Using the powerful technique of phage display, we
identified two peptides that show significant binding to the
brain compared to a control peptide. It is interesting to see
that two peptides with opposite net charges both bind to the
blood-brain barrier. Cell membranes are negatively charged
(44). Therefore, positively charged molecules may bind to the
cell surface non-specifically by electrostatic interactions (45).
Selected peptide GYR and control peptide RVR are both
positively charged (Table I), yet GYR shows a significantly
higher binding to the brain than RVR. Furthermore, the
negatively charged GLA shows significant binding to the
brain. These results indicate that the binding of the selected
Fig. 2. Incubation of phage with hCMEC/D3 cells, recovery
expressed as a percentage of the input. Mean±S.D.,n=3. A. Recovery
of phage after glycine wash (cell surface-bound phage). One way
ANOVA after log transformation to correct for non-Gaussian
distributions with Newman-Keuls correction for multiple comparisons
*p<0.05 GLAvs RVR and GYR vs YLR. **p<0.01 GYR vs RVR. B.
Recovery of phage after subsequent cell lysis (strongly bound/
internalized phage). One way ANOVA after log transformation with
Newman-Keuls correction for multiple comparisons **p<0.01 vs all
controls. Anterior vs posterior Mann-Whitney test p<0.01.
Input phage (TU)
Bound phage (TU)
Fig. 3. Dose-response curve of phage GLA, GYR and control phage
YLR. Mean±S.D., n=2. One way ANOVA after log transformation to
correct for non-Gaussian distributions with Dunnet’s correction for
multiple comparisons against control. GLA and GYR vs YLR p<0.01.
AUC is 14× and 26× higher for GLA and GYR, respectively.
678 van Rooy et al.
peptides is dependent on more than electrostatic interactions
with the endothelium.
When looking at the recovery from brain and lung, a
notable difference in recovery percentages was evident. The
base level of phage recovery from lung was higher than from
brain. This can be explained by mainly two factors. First,
traveling from heart to brain, the perfused fluid passes a
longer distance compared to perfusion through the lung.
Lungs are directly connected to the heart, allowing all of the
perfused fluid to reach the lungs. For reaching the brain,
fluids have to cover some distance, in which not all branching
vessels (i.e. subclavian arteries) could be clamped. This may
have resulted in increased loss of phage. Secondly, the total
surface area of the lung vasculature is larger than the total
brain surface area.
Identification of the peptide binding site can be an
important step in the ligand discovery process. This can give
insight into the ligand-target interaction and the uptake
mechanism. The binding site should preferably be a receptor,
which can specifically internalize a ligand, and the attached
drug delivery vehicle, into the cells. Alternatively, nano-
carriers such as liposomes can be delivered to transfer their
drug load to the target cells without the need for internal-
ization. Lipophilic drugs can be transferred from liposomes to
cell membranes when they are brought in close proximity by
a targeting ligand (46). This can result in higher uptake of
drugs with a low but not absent brain penetration capacity.
The GLA and GYR binding sites and potential uptake
mechanisms are as yet unknown.
Because of the negative selection that was performed by
perfusion through the heart, the binding site is likely to be
brain-endothelium specific. Therefore, it could be a new, yet
unidentified receptor, suitable for specific targeting to the
A literature search showed that 4 out of the 34 selected
peptides (PFA (47), GLD (48), SAY (49), and HAA (50))
had been selected before by other groups for very diverse
purposes. Two possible explanations could account for this
observation. These phage may have been selected in diverse
settings because they contain biologically relevant motifs,
suitable for multiple in vitro and in vivo applications.
Alternatively, these motifs may also just enhance phage
amplification rates, making them typical background phage
that do not offer a specific target interaction.
GLA GYR GTWYLR
Fig. 4. Incubation of selected phage GLA, GYR, GTW and control phage YLR with
hCMEC/D3 and HUVEC cells, recovery expressed as a percentage of the input. Mean±S.
D., n=3. A. Recovery of phage after glycine wash (cell surface-bound phage). B. Recovery
of phage after subsequent cell lysis (strongly bound/internalized phage).Students t-test
after log transformation to correct for non-Gaussian distributions. *p<0.05, **p<0.01
hCMEC/D3 vs HUVEC.
GLA GYR RVR
Fig. 5. Recovery of phage GLA, GYR, and control phage RVR from
whole murine brains after phage perfusion and washing. Recovery is
expressed as a percentage of the input. Mean±S.D., n=3. One way
ANOVA after log transformation to correct for non-Gaussian
distributions with Dunnet’s correction for multiple comparisons
against control. GLA and GYR vs RVR p<0.01.
Fig. 6. Recovery of phage GLA, GYR, and control phage RVR from
whole murine lungs after phage perfusion and washing. Recovery is
expressed as a percentage of the input. Mean±S.D., n=3. One way
ANOVA after log transformation to correct for non-Gaussian
distributions with Dunnet’s correction for multiple comparisons
against control. GLA vs RVR p<0.05.
679Peptide Ligands for BBB Targeting
Our selected peptides, as they have been tested here, are
expressed on a phage, displaying five peptides each. Attached
to a nanoparticle (e.g. liposome), the number of peptides per
particle can be greatly increased. This multivalency may
result in a stronger binding of the targeted nanoparticle (i.e.
avidity), compared to the phage tested here.
Two new peptides have been identified that may be used
for specific targeting to the blood-brain barrier. In the future,
these peptides could act as targeting ligands on nanoparticles
to enhance uptake of CNS drugs into the brain.
Fig. 7. Microscopic observation of hCMEC/D3 cells incubated with phage. A–D. Fluorescence microscopy
image of phage labeled with alexa-568 (red) and DAPI-stained cell nuclei (blue). A. No phage. B. Control
phage RVR. C. Selected phage GLA. D. Selected phage GYR. E–H. Light microscopy image of the same
view on the cell monolayer as the fluorescence image to the left.
680van Rooy et al.
We thank George P. Smith for providing the phage
display library and the host bacteria. This work was
supported by Top Institute Pharma, project T5-105-1: nano-
science as a tool for improving bioavailability and blood-brain
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which
permits any noncommercial use, distribution, and reproduction in
any medium, provided the original author(s) and source are
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