(783e) Characterizing Spions Permeability By Using An in Vitro Blood-Brain Barrier Model

Abstract: In the present study, using
murine brain endothelioma cells, an in vitro
blood-brain barrier model was set up and confirmed. Confirmation of the
blood-brain barrier model was completed by examining the permeability of
FITC-Dextran at increasing exposure times in serum-free medium and comparing
such values with values in the literature. After such confirmation, the
permeability of eleven different magnetic nanoparticle samples (SPIONs) was
determined by this blood-brain barrier model. Through these experiments, SPIONs
that would not pass through the blood brain barrier were identified for MRI
usage and SPIONs which would pass through the blood brain model were identified
for drug delivery usage.

Keywords:  nanoparticles,
blood-brain barrier, permeability
 Introduction

The blood-brain barrier is known as a "wall"
that separates somatic circulating blood from the cerebrospinal fluid in
central nervous system (CNS).
Consisting of tight junctions that lie between endothelial cells of capillaries
in CNS, it keeps large or hydrophilic molecules and microorganisms from passing
into the cerebrospinal fluid and therefore maintains the microenvironment. [1]

However, previous research shows that small
lipid-soluble-molecule that has a molecular weight less than 600 Da or, 200 nm
in diameter, can be transported through blood-brain
barrier. Therefore it
requires that nanoparticles, as effective pharmaceuticals, are able to undergo
successfully transportation through the blood-brain
barrier. [2]

Among several nanoparticles, superparamagnetic
iron oxide nanoparticles (SPIONs) have been discovered as a contrast agent which can potentially increase image contrast and improve
MRI sensitivity and specificity. Moreover, SPIONs can also be used to track
brain activity and detect cellular receptors that presented in brain diseases. On
the other hand, if the SPIONs inside blood-brain
barrier have not been efficiently cleared, the accumulation of those nanoparticles
could be toxic and harmful to normal brain function in a long term. [3]

Since there are concerns about both the application of
delivering drugs across the blood-brain
barrier and the accumulation of SPIONs in the body, this study set up an
in vitro blood-brain barrier
model to test new SPIONs' ability
to pass through blood-brain barrier.

Materials And Methods [4]

Material Characterization

According to a patented process entitled "A biomimetic
process for the synthesis of aqueous ferrofluids for
biomedical applications" [5], the synthesis of ferrofluids  involved incubation of a ferrous/ferric
salt solution in phosphate-buffered saline supplemented with the additives of
interest using ammonium hydroxide under highly alkaline conditions. After
synthesis, ferrofluids were centrifuged for a
stability test. Dynamic light scattering was used to characterize hydrodynamic
diameter and zeta potential was used to characterize the charge of each sample.
Confirmation of
the blood-brain barrier model

Characterization was carried out through fluorescent sugar
transport to confirm the blood-brain barrier model. For the blood-brain barrier model, murine
brain endothelioma cells (b.End3) were used, and
cultured in Dulbecco's modified
Eagle medium (DMEM) with 10% fetal bovine serum (FBS)
and 1% penicillin-streptomycin (P/S). In
the next step, 6.5 mm Transwell®-COL collagen-coated
0.4 µm pore polytetrafluoroethylene membrane inserts were combined with
24-well plates for the model. A hemocytometer and a
light microscope were used to determine the concentrations of cells. After
diluted down to 10⁵ cells per mL in DMEM + 10% FBS + 1% penicillin-streptomycin,
cells in the previous cell culture section were pipetted onto the inserts. Then
the well plates with inserts were incubated with daily changed medium in the
outer well until confluency was reached. After
reaching the confluency, medium should then be
replaced by 1:1 DMEM/Ham's F12
with 1% penicillin-streptomycin for the next 24, 48, 72 and 96 hours
respectively.

To confirm the blood-brain barrier model, the permeability
of the inserts would be tested in triplicate. After each time point was reached,
inserts should be transferred to wells that contain 600 µL
of Hank's Balanced Salt
Solution (HBSS) before adding fluorescein isothiocyanate-labeled
dextran (FITC-Dextran).
Immediately after the transfer, the permeability of the inserts was tested in triplicate using 10 µg/mL FITC-Dextran. Positive controls were set up by adding 100 µL
of FITC-dextran solution with 600 µL of HBSS and
negative controls were set up by adding 700 µL of HBSS only.

After 2 hours incubation, 100 µL
were taken from each well and transferred to a black 96-well plate with a clear
bottom. A fluorescent plate reader was excited at 490/20 nm and measured at
528/20 nm. Data were converted into relative fluorescence Unit (RFU) from the
reader and relative permeability was then determined:
Experimental
samples in the blood-brain barrier model

The previously described in vitro blood-brain barrier model was used to test the
permeability of the various nanoparticles, among which three of them were solid
powder (IONP) containing bovine serum albumin (BSA), polyvinyl alcohol (PVA),
collagen, and graphene and the remaining eight
samples were ferrofluids (FF) containing BSA,
collagen, graphene, glutamic acid, and four
concentrations (0.5%, 1.5%, 2.0%, and 2.5%, w/w) of PVA.

The inserts were transferred into a 24-well plate that
containing 600 µL
of HBSS in each well. Nanoparticles
were diluted 1:19 with HBSS to lower the concentration. Next, followed the
permeability experiment talked above, the inserts were exposed to the
nanoparticles for 2 hours. After 2 hours, 500 µL would be
pipetted from each well into a 20 mL scintillation vial and the inserts were
removed.

To determine their iron concentration, aqua regia was used to convert IONPs into ionic iron. Then
inductively-coupled plasma atomic emission spectroscopy (ICP-AES) was used to
determine the iron concentration.

Results and Discussion

        Material Characterization

The DLS results
showed that hydrodynamic diameters of nanoparticles range from 20 nm to 200 nm.
The results of zeta potential showed the lowest absolute zeta values for IONP
with collagen is -5.36 mV, for ferrofluids with PVA
and glutamic acid is -3.38 mV, and for ferrofluids
with collagen is -5.39 mV. The highest absolute zeta values were from IONP with
graphene and ferrofluids
with graphene.

Confirmation of
the blood-brain barrier model

The value of the experimental blood-brain barrier model
was in accordance with values in the previous articles, indicating that the
model was successfully established. First, the decline, which was about 30%, in
permeability was similar with the decline described by Brown et al. [6] Furthermore,
the decline of permeability with increasing exposure to serum-free medium also
indicated the success of a tightening of the barrier model.
Experimental
samples using blood-brain barrier model

Permeability of each sample has been tested and further experiments
were carried out to test whether one would be able to set up parameters to
moderate permeability of nanoparticles by using zeta potential, size, or
surface modification. Results showed that the highest permeability was obtained
from ferrofluids (FF) with collagen (1.5%
collagen-FF) and the lowest permeability was obtained from IONP with collagen
(1.5% collagen-IONP). These results suggest that for nanoparticles that need to
be delivered through blood-brain barrier, FF should be coated with collagen
and, on the other hand, IONP should be coated with collagen to keep
nanoparticles from penetrating the blood-brain barrier.

Conclusion

An in vitro model
of blood-brain barrier was established using b.End3 cells. As the permeability
decreased with increasing exposure to serum-free medium, the model was
confirmed by comparing the permeability trend of FITC-dextran in serum-free
medium with previous research. With the successfully established model, the
permeability of eleven nanoparticle samples was then carefully examined. Results
showed that in order to be delivered through blood-brain barrier, FF should be
coated with collagen and IONP should be coated with collagen to avoid
penetration.

Acknowledgment

The authors would like to thank the Northeastern
University for funding this research.

References

[1]     Hamilton RD, Foss AJ, Leach L (2007).
"Establishment of a human in vitro
model of the outer blood–retinal barrier". Journal of Anatomy 211 (6):
707-16.

[2]     Pardridge, W. M. (1995). "Transport of small molecules
through the blood-brain barrier: biology and methodology." Advanced Drug
Delivery Reviews 15(1-3): 5-36.

[3]     Berry CC, Curtis ASG. Functionalisation
of magnetic nanoparticles for application in biomedicine. J Phys
D Appl Phys. 2003; 36:198-206.

[4]     Dan Hoff, Lubna Sheikh,
Thomas J Webster. Comparison of ferrofluid and powder
iron oxide nanoparticle permeability across the blood-brain barrier.International
Journal of Nanomedicine. 2012; 2012:7-1.

[5]     Nayar S, Sinha A, Pramanick AK, inventors. A biomimetic process for the
synthesis of aqueous ferrofluids for biomedical
applications. Application number 0672DEL2010. March 22, 2010.

[6]     Brown RC, Morris AP, O'Neil RG. Tight junction protein
expression and barrier properties of immortalized mouse brain microvessel endothelial cells. Brain Res. 2007; 1130:17–30.