BLOOD-BRAIN BARRIER PERMEATION OF PACLITAXEL

Author : Aditya Grover, Anjali Hirani, Vijaykumar Sutariya

Page Nos : 71 - 78

Cite Article :

Grover A, Hirani A, Sutariya V. Blood-Brain Barrier Permeation of Paclitaxel. NUJPS. 2014;1(1):71–8.

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Introduction

Brain cancers, primarily glioblastomas,
present with poor prognoses in most
patients and have very high clinical
malignancies [1]. Central nervous system
(CNS) tumors are associated with a
number of undesirable side effects, namely
nausea and vomiting, headaches and
seizures [2]. Gliomas are mainly primary
tumors which arise from glial cells;
however, there are instances of
metastasized breast and lung cancers that
induce the formation of brain tumors [2,3].
Gliomas are classified into three main
categories by the World Health
Organization (WHO): astrocytomas,
oligodendrogliomas and oligoastrocytomas;
and each of them is graded
from I to IV with an increasing level of
malignancy [3]. Although, a number of
drugs express promising in-vitro efficiency
against brain cancers, the therapeutic
outcome of these drugs are observed to be
limited [4]. This may be due to the Blood
Brain Barrier (BBB), which develops
through the interactions of endothelial
cells, astrocytes and pericytes; resulting
into formation of tight junctions in the
endothelial cells, which prevents the entry
of the drug and other substance into brain.
BBB is the major hurdle for variety of anticancer
drug in the treatment of brain
cancer

Paclitaxel is proven potent anti-cancer
drug for brain tumor; however, the
permeability through the blood brain
barrier is the rate limiting parameter for
paclitaxel drug delivery. Paclitaxel is
commercially administered as Taxol,

belongs to the taxane family of drugs,
which is derived from the bark of the
pacific yew tree (Taxus brevifolia) and has
been clinically used against a number of
cancers including ovarian and breast
cancers and particularly significantly
active against malignant gliomas and
metastasis [1,2,5,7]. Paclitaxel has a
unique mechanism of action that it
promotes the assembly of microtubule
which in turn prevents depolarization for
cell division in the G2 or M phase of cell
mitosis causing the cell death [3].
However, specific structural property of
brain anatomy and limited permeability of
paclitaxel reduces the access of drug at
brain tumor. Additionally, Paclitaxel
structure is also the subtract for multidrug
resistant protein, which express pglycoprotein
(p-gp). Such expressed
proteins like p-gp transporter efflux-out the
paclitaxel with their transport mechanism
[7, 8]. Hence, in conclusion, variety of
research it has been proved that
permeability of paclitaxel in brine is
limiting parameter for brain cancer
therapy. According to Fellner et al [6] 90
% of brain tumor was observed to be
reduced by administration of paclitaxel
with p-gp inhibitor as compare to
paclitaxel alone.

The poor aqueous solubility of paclitaxel
due to macro-molecular structure, limits
the development of conventional
intravenous injections to improve
bioavailability at site of action. To improve
the solubility of paclitaxel, the co-solvency
approach have been explored, whereby 1:1
volume ratio of Cremophor EL and ethanol
has shown improved pharmaceutical

perspectives of formulation [4]. However,
cremophor EL has been shown to cause
hypersensitivity reaction, which limits the
clinical application. Although, paclitaxel
has been successfully used in a number of
different cancers, its use in brain cancers
has been limited due to the blood-brain
barrier (BBB), which impedes the
permeation of possible toxins into neural
tissue. To surmount the BBB’s challenge to
brain-targeted drug delivery, many
attempted has been like liposomes,
nanoparticles, micelles and conjugates etc.
[9]. After variety of attempts, few
formulation (i.e. Xyotax) has been proved
as a promising against in phase-II and
phase-III clinical trials for brain metastasis
[10]. However, the review from the
scientist does not expect its efficiency to
deliver paclitaxel in to the brain for brain
cancer. Therefore, the in-depth research is
still required to deliver paclitaxel in brain
with high efficiency. The Aim of this
present work is to develop efficient
carriers, which are able to deliver anticancer
drug in to the brain for brain cancer
therapy.

In present investigation, glutathione was
explored as a coating material for efficient
delivery of paclitaxel nanoparticle in to the
brain for brain cancer. Out of number of
mechanisms proposed to induce the trans-
BBB permeation of drugs, a glutathione
method has shown success in in-vitro and
in-vivo studies [11, 12]. Glutathione is the
substrate for p-gp protein which is express
in brain and therefore the nanoparticles
conjugated with glutathione may be able to
cross BBB. Therefore, it was predicted that
conjugation of glutathione with

nanoparticles loaded with methylene blue
as well as nanoparticles loaded with
coumarin may improve the permeation of
drug through BBB.
Material and method

Material

PLGA was supplied by CHS pvt. Ltd.,
Acetone and Poloxamer 188 were
purchased from sigma eldritch, EDAC
was procured from MP Biomedical.
Glutathione, Coumarine, Methylene blue
were supplied by Venus Chemicals.
Methanol used was of analytical grade.
Deionized water used for further
experiments was obtained from Millipore.

Preparation of methylene blue loaded
nanoparticles

The methylene blue loaded PLGA
nanoparticles were prepared by
nanoprecipetation method proposed by
Patel N et al. [13] Initially, the PLGA was
dissolved in acetone with pluronic F-108.
Methylene blue was was separately mixed
with acetone using magnetic stirrer.
Organic solvent containing PLGA,
pluronic F-108 and methylene blue were
added drop wise at the rate of 2ml/min into
the deionized water under continuous
stirring at 900 RPM on magnetic stirrer.
The organic solvent was allowed to
evaporate at room temperature on constant
stirrer, resulting in formation of methylene
blue loaded nanoparticles of PLGA.
Further, the dispersion was centrifuge at
5000 RPM to separate the nanoparticles
from the suspension and were lyophilized
to obtain free flowing powder.

Preparation of coumarin loaded
nanoparticles

The coumarin loaded PLGA nanoparticles
were prepared by method proposed by
Vanya B et al. [14] The 1:10 proportion of
coumarin: PLGA were dissolved in 5 mL
of acetone. The solution was mixed with
10mL of aqueous poloxamer 188 solution.
The resulting suspension was stirred
alongwith heating at 60°C using a
magnetic stirrer for 3 hours to remove the
organic solvent from dispersion. Further,
the suspension was filtrated through a
“white tape” paper filter and lyophilized to
get free flow powder.

Conjugation of glutathione with
nanoparticles

Conjugation of glutathione with
nanoparticles was carried out as per the
methodology proposed by Acharya S et al
[15]; whereby 60 mg of nanoparticles were
added in 2.4 mL phosphate buffer (pH 4.0)
followed by drop-wise addition of an equal
volume of EDAC solution having
concentration of 50 mg/mL. The resultant
mixture was incubated at room
temperature for 45 minutes. The un-reacted
EDAC was removed by centrifugation and
the nanoparticles were re-suspended in 2.4
mL phosphate buffer (pH 7.4). The same
volume of glutathione solution having the
concentration of 50 mg/mL was added to
the dispersion containing the activated
nanoparticles and the mixture was stirred
gently at room temperature for 6 hours.
The conjugated nanoparticles were
centrifuged for 30 minutes at 15000 RPM
to remove excess of glutathione. Finally
the glutathione conjugated nanoparticles

were lyophilized to get free flowing
powder. Nanoparticles were prepared using
a nanoprecipitation method and were
coated with enough reduced glutathione to
yield a 2% w/v coating on the
nanoparticles.
Particle size measurement of nanoparticles
Particle size and particle size distribution
was measured by Malvern’s zeta-sizer. The
nanoparticles were dispersed in suitable
solvent and sonicated to prevent the
agglomeration. Later on, the particle size
was characterized after appropriate
dilutions with deionized water.
In-vitro study
The in-vitro permeation of glutathione
conjugated nanoparticles of PLGA was
performed through establishing a Transwell
co-culture of C6 (rat astrocytoma)
cells and RBE4 (rat brain endothelial) cells
[12] as shown in Figure 1, whereby the cell
culture was allowed to developed on both
the sides of permeable support.
The comparative trans-BBB permeation of
10 μM each of MB free drug solution,
uncoated MB nanoparticle and 2%
glutathione-coated MB nanoparticle was

Representation of the Transwell apparatus set-up to investigate the in-vitro permeability

Figure 1: Representation of the Transwell
apparatus set-up to investigate the
in-vitro permeability.

The comparative trans-BBB permeation of
10 μM each of MB free drug solution,
uncoated MB nanoparticle and 2%
glutathione-coated MB nanoparticle was

carried out by taking readings at
predetermined intervals (0, 3, 6 and 24
hours).
Results

PLGA Nanoparticles

Experimental variations like process
related parameters (i.e. stirring speed, rate
of addition, evaporation rate, evaporation
temperature, centrifugal force, incubation
time, etc.) as well as the formulation
parameters (i.e. concentration of polymers;
type, volume and ratio of organic solvent;
etc.) have shown to have the significant
effect on the formulation and development
of nanoparticles.

Particle Size measurement
Mean diameter of glutathione conjugated
methylene blue loaded nanoparticle was
found to be 220 nm, whereas glutathione
conjugated coumarin loaded nanoparticle
was 250 nm. Moreover, the particles size
distribution for the glutathione conjugated
methylene blue loaded nanoparticle was
ranging between 190 mm to 250 nm,
whereas glutathione conjugated coumarin
loaded nanoparticle was between 200 nm
to 300 nm.

In-vitro study

In-vitro study shown that the glutathione
conjugated nanoparticles showed a
significant increase in their trans-BBB
permeation over 24 hours, while the free
drug solution showed almost nil
permeation across the Transwell system
(Figure 2). Similar result pattern was
observed for both the types of
nanoparticles.

Trans-BBB permeation of MB drug solution as compared to MBloaded, after 24 hours of treatment in Transwell apparatus.
Figure 2: Trans-BBB permeation of MB
drug solution as compared to MBloaded,
glutathione-coated nanoparticles
after 24 hours of treatment in Transwell
apparatus.
Discussion
PLGA Nanoparticles
The results revealed that formulation of
nanoparticle is very complicated process
and variety of process related parameters
and the formulation related parameters
significantly affects the physico-chemical
properties of nanoparticles, which in turn
affect the therapeutic outcome of the
nanoparticles. Hence, careful design and
optimization of each variable is essential to
achieve desired properties of nanoparticles.
Particle size measurement
Experimental variables like amount of
polymer, amount of surfactant content and
amount of drug affect significantly to
particle size distribution. The amount of

PLGA content is directly proportional to
particle size diameter. According to
Quintanar Guerrero et al. [16] study of
PLGA content influence on nanoparticle
size distribution, which suggest that as
higher polymer amount promotes the
aggregation due to less repulsive force
between two particles, leading to increase
in the mean particle size by aggregating
with each other. The drug loading also
showed direct relation with particle size
distribution, due to higher entrapment
efficiency of carrier and free drug tries to
absorb on surface of nanoparticle causing
aggregate [17]. Glutathione content also
increases bulkiness of particle after
conjugation, therefore, over-time of
conjugation increases particle size [18].
In-vitro study

The in-vitro study of the glutathione
conjugated coumarin loaded nanoparticles
shown significant tran-BBB permeation as
compare to unconjugated nanoparticle,
which suggest that p-gp transporter effluxout
unconjugated nanoparticle
successfully; however, the glutathione
conjugated nanoparticles are able to cross
the BBB. Overall uptake study reveals the
vital role of glutathione as a carrier for
trans-BBB permeation. Therefore
glutathione may serve as an better vehicle
for paclitaxel to deliver it in brain for the
cancer treatment.

Conclusion

The in-vitro permeation study reveals that
the glutathione conjugated nanoparticles of
PLGA are able to cross the BBB. The
success of this glutathione method should

be investigated first in-vivo in animal study
and then in human subjects to determine its
clinical efficacy. This newer approach may
provide a valuable therapeutic outcome
and may provide the biomedical benefits in
the number of therapies that are targeted to
the brain and facilitates reduction of the
morbidity faced by patients with CNS
malignancies.
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