Journal of Biomaterials and Nanobiotechnology, 2011, 2, 485-493
doi:10.4236/jbnb.2011.225059 Published Online December 2011 (http://www.scirp.org/journal/jbnb)
485
Evaluation of Biotinylated PAMAM Dendrimer
Toxicity in Models of the Blood Brain Barrier:
A Biophysical and Cellular Approach
Heather A. Bullen1, Ruth Hemmer2, Anthony Haskamp1, Chevelle Cason1, Stephen Wall1,
Robert Spaulding2, Brett Rossow2, Michael Hester2, Megan Caroway2, Kristi L. Haik2
1Department of Chemistry, Northern Kentucky University, Highland Heights, USA; 2Department of Biological Sciences, Northern
Kentucky University, Highland Heights, USA.
Received October 12th, 2011; revised November 17th, 2011; accepted November 26th, 2011.
ABSTRACT
The interaction of biotinylated G4 poly (amidoamine) (PAMAM) dendrimer conjugates and G4 PAMAM dendrimers
with in vitro models of the blood brain barrier (BBB) was evaluated using Langmuir Blodgett monolayer techniques,
atomic force microscopy (AFM) and lactate dehydrogenase measures of cell membrane toxicity. Results indicate that
both G4 and G4 biotinylated PAMAM dendrimers disrupt the composition of the liquid condensed (LC) and liquid ex-
panded (LE) phases of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid monolayer. The disruption is
concentration dependent and more marked for G4 biotinylated PAMAMs. Lactate dehydrogenase (LDH) assays using
endothelial cell culture models of the BBB indicate that biotinylation results in higher levels of toxicity than non-bioti-
nylation. This approach p ro vides valuable information to assess nanoparticle toxicity for drug delivery to the brain.
Keywords: Dendrimers, Drug Delivery, Blood Brain Barrier, Toxicity
1. Introduction
Central nervous system (CNS) diseases, disorders and
injuries affect over 1.5 billion people worldwide and di-
agnosis and treatment of CNS disorders represent a con-
siderable challenge. The brain is separated from the rest
of the body by the blood brain barrier (BBB) and the
protective mechanisms of the BBB renders the brain a
site of poor permeability of various drugs and delivery
systems. The BBB is a tight seal of cells that lines the
blood vessels in the brain. The walls of BBB capillaries
are composed of specialized endothelial cells, brain cap-
illary endothelial cells (BCEC), which form tight junc-
tions. Tight junctions contain integral membrane proteins
that form a seal between adjacent endothelial cells. In
addition, accessory structures that surround the BCECs
include pericytes and associated astrocytes and neurons
[1-3]. The complexity of the BBB comes from the vari-
ety and number of transporters within the BBB, including
p-glycoprotein-1, glucose-related transporters, nucleoside
transporters, receptors for transferrin, insulin, leptin,
lectins, IGFs, various ATPases and many, many more
that are taken up via receptor mediated endocytosis [4].
This complexity also makes developing in vitro models
of the BBB challenging.
While the BBB is essential for maintaining a healthy
brain, it impedes efforts to deliver therapeutic agents into
the brain. The poor permeability of various drugs as well
as delivery systems across the BBB is primarily due to
tight junctions, lack of capillary fenestrations and pres-
ence of efflux transporters. The BBB can reportedly
block more than 98% of CNS drugs [5]. Due to the inef-
fectiveness of conventional drug therapies, finding ways
to deliver therapeutic drugs to the CNS safely and effec-
tively is essential. The development of novel strategies
that could overcome the obstacles of brain drug delivery
is essential. The application of nanoscience to CNS dis-
orders is an active area of research. A number of nano-
particle delivery systems have been developed and dem-
onstrated promising properties [5-8].
Dendrimers are appealing choices for nanoparticle
drug delivery because of the ability to control their pre-
cise architecture, size and shape, high uniformity and
purity, high loading capacity, low toxicity and low im-
munogenicity [9-12]. The presence of a large number of

Evaluation of Biotinylated PAMAM Dendrimer Toxicity in Models of the Blood Brain Barrier: 486
A Biophysical and Cellular Approach
surface groups provides opportunity to conjugate ligands
not only for transport across the BBB but also for target-
ing specific cells, such as tumors. Dendrimers can be
prepared with specific surface modifications that enable
the dendrimers to gain entry through a membrane while
holding a molecule that cannot pass on its own. Once the
dendrimer passes the membrane, it can deliver the drug
held in its interior.
There are a tremendous number of potential applica-
tions for dendrimers in biological systems [13-18], with
poly(amidoamine) PAMAM dendrimers being the most
widely studied [11] However, the application of den-
drimers in brain delivery is a relatively new area of re-
search. Several targeted drug delivery systems using
various targeting ligands have been used with some suc-
cess in terms of BBB crossing [5,8], including lactoferrin
[19], epidermal growth factors [20] and doxorubicin [21].
The mechanism of uptake and toxicity to the BBB has
not been extensively studied. A detailed characterization
of dendrimer toxicity is important for the design and use
of dendrimers in brain drug delivery. Toxicity of both the
functional group and generation of the dendrimer must
be taken into consideration. PAMAM dendrimers have
been shown to be haemolytic and cytotoxic, with toxicity
tending to be higher for cationic PAMAM dendrimers
and to increase with generation [22,23].
In this study we evaluated the potential toxicity of
biotinylated G4 PAMAM dendrimer conjugates. Biotin is
an important molecule used in several metabolic path-
ways and belongs to a family of molecules that have
been shown to cross the BBB [24,25]. Biotin-labeled
dendrimers have been utilized in tumor [26] and antibody
[27] targeting studies and biosensor design [28]. Bioti-
nylated PAMAM dendrimers may also have the potential
for delivering therapeutic drugs to the brain [24,29].
The biophysical interactions of biotinylated G4
PAMAM conjugates and G4 PAMAMs with lipid model
membranes were evaluated using Langmuir Blodgett
monolayer techniques and atomic force microscopy
(AFM). Results were correlated with cellular toxicity
measurements using endothelial cell culture models of
the BBB. This work reports the first analysis of PAMAM
dendrimers using this combined approach. The results
provide important insights into strategies for developing
nanoparticle systems for brain drug delivery.
2. Experimental Section
2.1. Materials
Poly(amidoamine) PAMAM dendrimers [core: ethylene
diamine]; (G = 4); dendri-PAMAM-(NH2)32) were ob-
tained from Dendritic Nanotechnologies, Inc. (Mt. Plea-
sant, MI). Biotinylated PAMAMs were prepared using
sulfo-NHS-LC-biotin (Pierce EZ-Link® Kit) as de-
scribed previously [30]. Biotinylated dendrimers were
resuspended (1.0 mg/mL) in 1.0 M phosphate buffer sa-
line (PBS) until used.
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
was obtained from Avanti Lipids (Alabaster, AL). All
organic solvents used were analytical, HPLC grade, from
Sigma (Sigma-Aldrich, St. Louis, MO). DI water was
obtained using a Milli-Q plus water purification system
(Millipore, Bedford, MA, USA). PBS and Borate buffers
were prepared from Pierce buffer packs (Pierce Protein
Research Products; Rockford, IL).
2.2. Langmuir Trough
A KSV 2000 Langmuir-Blodgett trough (KSV, Helsinki)
was used for lipid monolayer experiments. Interfacial
pressures were measured with a Wilhelmy balance using
a platinum plate and an aqueous subphase, DI water, was
used for all experiments. Before each experiment the
trough was cleaned twice with 200-proof ethanol and DI
water. All impurities remaining on surface were removed
by aspirating the subphase surface. To ensure the trough
was clean, the stability of the surface potential was
checked before each experiment. Lipid and dendrimer
solutions were prepared (0.5 mg/mL) in HPLC grade
chloroform immediately prior to use. Lipid/dendrimer
solutions were prepared by mixing dendrimers with the
DPPC solution (0.5 mg/mL) corresponding to various
lipid/dendrimer ratios (v/v). Monolayers were spread (60
μL of lipid or 60 μL lipid/dendrimer mixtures) onto clean
subphase (25˚C ± 1˚C) using a Hamilton syringe. After
evaporation of solvent (20 min) they were compressed as
a rate of 5.5 mm/min. For monolayer transfer experi-
ments a cleaved mica substrate was submerged into the
subphase prior to addition of lipids. The lipid monolayer
was spread as before and compressed to a desired surface
pressure. The mica was then withdrawn from the sub-
phase vertically a pulling rate of 5.5 mm/min once target
surface pressure had been reached.
2.3. AFM Analysis of Monolayers
A Dimension 3100 Digital Instruments SPM was utilized
in tapping mode (NSC 14 tip-140 Hz) for all image
analysis. Typical scan rates were 1 Hz and all images
were acquired in air. Lipid monolayers were prepared by
Langmuir Blodgett techniques, described above and were
prepared on freshly cleaved mica substrates. Samples
were stored in sealed containers and imaged immediately
after film deposition. Multiple analyses of transferred
monolayers was conducted.

Evaluation of Biotinylated PAMAM Dendrimer Toxicity in Models of the Blood Brain Barrier: 487
A Biophysical and Cellular Approach
2.4. bEnd.3 Cell Culture
A murine brain capillary endothelial cell line, bEnd.3,
was purchased from American Type Culture Collection
(ATCC, CRL-2299) and cultured as recommended by the
supplier. Briefly, bEnd.3 cells were cultured in media
containing Dulbecco’s Modified Eagle’s Medium (DMEM;
ATCC, 30 - 2002) containing 4 mM L-glutamine, 4500
mg/L glucose, 1 mM sodium pyruvate, 1500 mg/L so-
dium bicarbonate and supplemented with penicillin (500
u/mL), streptomycin (100 μg/mL) and 10% fetal bovine
serum (FBS) in a humidified 5% CO2, 95% air incubator
at 37˚C. At 80% confluency, cells were trypsinized,
counted and seeded in 96-well flat-bottomed plates with
a density of 1.7 × 105 cells/well. After the cells grew for
2 days, cells were rinsed once with media and cells were
then treated with one of the following treatment groups:
3 μL of 1 mg/mL G4 biotinylated dendrimers; 3 μL of 1
mg/mL G4 dendrimers; 3 μL of culture media alone; or 3
μL of 0.05% sodium azide (each added to 197 μL of me-
dia). After 24 h of treatment (in humidified 5% CO2,
95% air incubator at 37˚C) the media was collected and
used for LDH analysis. Fresh media was added to wells
and after 24 h media was collected again for LDH analy-
sis. Cell phenotype was confirmed by an immunocyto-
chemical stain for von Willebrand factor [31].
2.5. Lactate Dehydrogenase Assay (LDH)
Mouse cerebral endothelial cell death was quantitatively
assessed using the Tox-7 LDH based in vitro toxicology
assay kit (Sigma). An LDH assay was performed on me-
dia collected after 24 and 48 h. LDH values were com-
pared between control and treatment conditions using a
one-way ANOVA (SPSS) and Bonferroni post-hoc
analyses when appropriate.
3. Results and Discussion
3.1. Compression Isotherms
Compression isotherms provide information on if
PAMAM dendrimers penetrate into the lipid monolayer
and influence lipid organization. The surface pressure-
area isotherms (π-A isotherms) for DPPC and DPPC with
G4 PAMAM and G4 biotinylated PAMAM dendrimers
are shown in Figure 1. The isotherm for pure DPPC is
similar to that reported in the literature [32,33] and
shows the well-known phase transition from liquid-ex-
panded (LE) to liquid condensed (LC) phases at around
10 mN/m. Addition of G4 and G4 biotinylated PAMAM
dendrimers leads to a slight expansion in the disordered
LE phase of the DPPC monolayer and the formation of
an indistinct phase transition region. Increasing amounts
of G4 and G4 biotinylated PAMAM dendrimers seem to
have a greater effect on the disappearance of the DPPC
LE-LC phase transition. The PAMAM dendrimers also
affected the collapse pressure of the DPPC monolayer
(55 mN/m); in general, higher concentrations of PAMAM
dendrimers lead to a lower collapse surface pressure.
This disruption of lipid monolayer stability is more pro-
nounced for G4 biotinylated PAMAM dendrimers (Fig-
ure 1(B)).
These results indicate that both G4 and G4 bioti-
nylated PAMAM dendrimers are initially incorporated
into the DPPC monolayer. This incorporation disturbs
the organization of the DPPC monolayer, as evident by
(A)
(B)
Figure 1. Surface pressure vs. area isotherm of DPPC lipid
monolayers in the presence of different concentrations (v/v)
of (A) G4 PAMAM dendrimers and (B) G4 biotinylated
PAMAM dendrimers.

Evaluation of Biotinylated PAMAM Dendrimer Toxicity in Models of the Blood Brain Barrier: 488
A Biophysical and Cellular Approach
the shift of the overall isotherm curve to lower molecular
areas, loss of the LE-LC phase transition region and de-
crease of the surface collapse pressure. The PAMAM
dendrimers fluidize the monolayer at low surface pres-
sures, causing more lipid molecules to exist in the LE
state rather than the LC phase [34-36]. Upon compres-
sion an enhanced squeeze out of material into the sub-
phase is observed for both DPPC/G4 and DPPC/G4
biotinylated PAMAM dendrimer mixtures, as indicated
by the shift of the isotherms to lower molecular areas and
the kink evident at ~6 mN/m [34,36]. Previous research
suggests that PAMAM dendrimers have an electrostatic
nature of binding to lipid membranes [37], implying that
primarily the polar head groups of DPPC and the surface
amino groups of the G4 PAMAM dendrimers are in-
volved in the interaction. This interaction would slightly
affect the packing of the acyl chain domain, leading to
the area expansion evident in the LE phase [38]. The
comparable increase in area expansion in the LE region
suggests that the G4 biotinylated PAMAM dendrimers
affect the packing density of DPPC in a similar fashion.
3.2. AFM Analysis
AFM is a direct imaging tool for visualizing the lipid
monolayers to determine the influence of G4 and G4
biotinylated PAMAM dendrimers on changes in the mi-
crodomain formation of the LE and LC phases. The
AFM analysis of pure DPPC monolayer deposited at 12
mN/m is shown in Figure 2. At this surface pressure, the
LE and LC phases coexist, with the bright regions corre-
sponding to the LC phase (a more upright configuration)
and the dark regions corresponding to the LE phase. The
LC phase domains are ~2.2 ± 0.6 µm long and ~124 ± 35
nm wide. These domains are separated by regions of the
LE phase (gap is on average ~200 nm). Within the LE
Figure 2. AFM height analysis of DPPC lipid monolayer (12
mN/m).
phase region smaller islands of LC phase are also evident
(~30 - 40 nm in diameter).
The influence of G4 and G4 biotinylated PAMAM
dendrimers on the DPPC domain structure is shown in
Figures 3 and 4. Increasing concentrations of G4 PAM-
AM dendrimers reduce the formation of the LC phase
domains. At low G4 PAMAM dendrimer concentrations,
DPPC:G4, 2:1 (Figure 3(A)), some regions look similar
to DPPC alone, with long “stripe-like” LC phase do-
mains (~1.0 ± 0.2 µm long; ~68 ± 29 nm wide) separated
by ~150 - 200 nm LE phase domains. However other
regions show a more significant change in LC phase do-
main size, as evident by a more “oval, island-like” struc-
tures (~95 ± 20 nm diameter) separated by ~50 nm LE
phase domains. Upon increasing concentration of G4
PAMAM dendrimers (DPPC:G4, 1:1), the “oval, is-
land-like” LC phase regions become more prevalent
within the mono- layer (Figure 3(B)). The LC phase
islands (~150 ± 45 nm in diameter) are separated by ~50
nm LE phase domains. In addition a “honeycomb-like”
LC phase structure is also evident, exhibiting larger re-
gions of LE phase within the monolayer. Under higher
concentrations of G4 PAMAM dendrimers (DPPC:G4,
1:2) two different domain structures are apparent, each
leading to a further increase in the LE phase composition
(Figures 3(C) and 3(D)). Some regions of the monolayer
(Figure 3(C)) show LC phase islands that are quite var-
ied in shape (roughly ~500 nm diameter) separated by
LE phase regions (~300 - 400 nm gaps), which make up
~68% of the monolayer composition. Other regions show
further in- creases in LE phase composition (Figure 3(D))
with the LC phase existing as lines (~720 ± 180 nm long;
~31 ± 4 nm wide) separated by LE phase regions (~300-
350 nm gaps), which make up ~80% of the monolayer
composition.
Low concentrations of G4 biotinylated PAMAM den-
drimers, DPPC:G4bio, 2:1 (Figure 4(A)), cause an ag-
gregation of the LC phase to form larger stripes com-
pared to those found in DPPC alone. The LC and LE
phase domains coexist as alternating stripes (>3 µm long;
~273 ± 86 nm wide); within the LE regions, small islands
(~35 - 50 nm diameter) of the LC phase also exist. Upon
increasing concentration of G4 biotinylated PAMAM
dendrimers (DPPC:G4bio, 1:1), two different domain
structures are evident (Figures 4(B) and 4(C)), both
showing an increase in the LE phase composition of the
monolayer. Figure 4(B) shows LC phase stripe domains
(~800 ± 150 nm long, ~210 ± 80 nm wide) separated by
LE phase regions (~400 nm - 1.5 µm gaps), which make
up ~80% of the monolayer composition. Figure 4(C)
shows LC islands domains that vary in shape (roughly 1
µm in diameter) separated byhase regions (~350 nm LE p

Evaluation of Biotinylated PAMAM Dendrimer Toxicity in Models of the Blood Brain Barrier:
A Biophysical and Cellular Approach
489
Figure 3. AFM height analysis of DPPC lipid monolayers (12 mN/m) in the presence of different concentrations (v/v) of G4
PAMAM dendrimers: (A) DPPC:G4, 2:1; (B) DPPC:G4, 1 : 1; (C) DPPC:G4, 1:2; (D) DPPC:G4, 1:2.
- 600 nm gaps), which makeup ~65% of the monolayer
composition. Under higher concentrations of G4 bioti-
nylated PAMAM dendrimers (DPPC:G4bio, 1:2) a fur-
ther increase in the LE phase region is evident (Figure
4D). The LC phase exists as lines (~716 ± 210 nm long;
~24 ± 6 nm wide) separated by LE phase regions (~250
nm gaps). Larger regions of LE phase (1 - 2 μm) are also
evident, with the LE phase making up ~89% of the
mono- layer composition.
AFM analysis reveals that both G4 and G4 bioti-
nylated PAMAM dendrimer conjugates disrupt the com-
position of the LC and LE phases of DPPC. The disrupt-
tion is concentration dependent and more marked for G4
biotinylated PAMAM dendrimers. These findings are in
agreement with compression isotherm measurements,
which showed a change in the LE-LC phase transition
and decrease in collapse pressure, reflecting a clear de-
stabilization of the DPPC packing upon addition of the
PAMAM dendrimers. AFM analysis showed no evidence
of PAMAM dendrimers within the monolayers. This is
also in agreement with compression isotherm analysis
which indicated that there was a squeeze out of material
from the monolayer upon compression to higher surface
pressures. The occupation of lower molecular areas for
the isotherm suggests that in addition to the PAMAM
dendrimers, lipids may also have been expelled from the
monolayer. One possible mechanism could be that strong
lipid-polymer interactions cause the DPPC molecules to
surround the dendrimers, forming vesicles in solution
that escape into the subphase [39].
3.3. Cell Toxicity
The bEnd.3 immortalized endothelial cell line was cho-
sen as a basic model of the BBB. Endothelial cells make
up a majority of the BBB, which controls cerebral ho-
meostasis and prevents toxins and other chemicals in the
blood stream from entering the brain. Therefore, it is
vital to understand how nanoparticles affect these cells.
The endothelial phenotype of the bEnd.3 cell line was
confirmed by the observed expression of von Willebrand
factor and uptake of fluorescently labeled low density
lipoprotein (LDL) [31].
To obtain a specific measure of toxicity, the LDH as-
say was used for spectrophotometric measurement of

Evaluation of Biotinylated PAMAM Dendrimer Toxicity in Models of the Blood Brain Barrier: 490
A Biophysical and Cellular Approach
Figure 4. AFM height analysis of DPPC lipid monolayers (12 mN/m) in the presence of different concentrations (v/v) of G4
biotinylated PAMAM dendrimers: (A) DPPC:G4bio, 2:1; (B) DPPC:G4bio, 1:1; (C) DPPC:G4bio, 1:1; (D) DPPC:G4bio, 1:2.
viable cells (Figure 5). LDH is a soluble cytosolic en-
zyme that is released into the culture medium following
loss of membrane integrity resulting from either apop-
tosis or necrosis. LDH activity is commonly used as an
indicator of cell membrane integrity and serves as a gen-
eral means to assess cytotoxicity resulting from chemical
compounds or environmental toxic factors. Briefly, LDH
reduces NAD+, which then converts a tetrazolium dye to
a soluble, colored formazan derivative. The absorbance
of the converted dye is measured at a wavelength of 490
nm [40-42]. Therefore, the higher the absorbance, the
more cell damage. While certain nanoparticles have been
shown to inactivate LDH, producing false negative toxic-
ity results [43], this is not a concern with the dendrimers
in the study.
As shown in Figure 5, bEnd.3 cells were exposed to
one of the following treatment conditions: G4 bioti-
nylated PAMAM dendrimers; G4 PAMAM dendrimers;
culture media alone (negative control); or sodium azide
(positive control). After 24 h no significant differences
were detected in the amount of LDH produced by the
cells across the four treatment groups [F(3,11) = 2.892, p
> 0.05]. However, after 48 h, cells produced significantly
more LDH [F(3,11) = 40.015, p < 0.001]. Post-hoc
analyses show that both types of dendrimers and sodium
azide were more toxic than media alone. Additionally,
biotinylated dendrimers were more toxic to the cells than
any of the other treatment groups. These findings suggest
that biotinylation of G4 PAMAM dendrimers results in
higher levels of toxicity than non-biotinylation in this
cell culture system. Cell toxicity measurements are in
Figure 5. LDH activity of bEND.3 cells exposed to G4
biotinylated PAMAM dendrimers, G4 PAMAM dendri-
mers, or sodium azide. Values represent mean ± SEM. *p <
0.001 relative to media alone, dendrimers and sodium azide;
**p < 0.001 relative to media alone.

Evaluation of Biotinylated PAMAM Dendrimer Toxicity in Models of the Blood Brain Barrier: 491
A Biophysical and Cellular Approach
agreement with Langmuir compression isotherms and
AFM analyses. Although it is important to note that the
dosage of dendrimers may impact toxicity measurements
[44].
4. Conclusions
The findings presented here show the potential toxicity
of G4 and G4 biotinylated PAMAM dendrimers and how
biophysical measurements with model lipid systems can
be correlated with cell toxicity analysis to provide infor-
mation on nanoparticle toxicity. At this time the exact
factors that lead to the increased toxicity measured for
biotinylated PAMAM dendrimers are unknown. How-
ever, controlling the degree of surface functionalization
could potentially reduce dendrimer toxicity [22] (currently
~17% surface coverage) [30]. It is possible that biotiny-
lated PAMAM dendrimers may prove to be more toxic
compared to PAMAM dendrimers alone, in part, due to
their potential mechanism of uptake across the BBB, as
biotin has shown to cross the BBB through carrier me-
diated endocytosis [24,25]. Therefore, there may be more
biotinylated PAMAM dendrimer conjugates getting into
cells compared to non-biotinylated PAMAM dendrimers,
leading to more cell death. Although it is important to
note that the dosage of dendrimers may impact toxicity
measurements [44].
5. Acknowledgements
This project has been funded by the Merck Institute for
Science Education, Kentucky NSF EPSCoR, Northern
Kentucky University, Center for Integrated Natural Sci-
ence and Mathematics and the NKU Research Founda-
tion. This work was also supported by a grant from the
Kentucky Biomedical Research Infrastructure Network
(P20 RR016481-08) and the National Science Founda-
tion (DMR-0526686, CHE-0619342).
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