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Delivery Systems
Gene Delivery Across
the Blood Brain Barrier
Nanoparticle technology is being used to facilitate the delivery of
biopharmaceutical compounds across the blood brain barrier, opening up
new possibilities for the treatment of brain disorders that would previously
have been inconceivable.
By Kerstin Ringe at NanoDel Technologies GmbH
Dr Kerstin Ringe obtained a PhD in synthetic organic chemistry from the Technische Universität, Braunschweig, Germany,
in 1995. She then joined the group of Professor Okamura at the University of California, Riverside, focusing on Vitamin
D3 chemistry. Over the period 1997-99, she was involved in research on Epothilon Derivatives. In 2000, Dr Ringe
headed the laboratory of Product and Process Development at NanoPharm AG (Magdeburg, Germany) and, since 2004,
has been responsible for Patent and Project Management at NanoDel Technologies GmbH in Magdeburg.
The role of the blood brain barrier (BBB) is to
protect the brain against toxic substances that
circulate in the bloodstream. Although the BBB is a
fairly ‘tight’ structure when ‘unfriendly’ molecules try
to enter the brain, it does have several mechanisms
to allow ‘friendly’ molecules to get in. Among them
are active, carrier-mediated transport mechanisms for
relatively small molecules, an absorptive-mediated
endocytosis mechanism for positively charged
peptides and receptor-mediated endocytosis
mechanisms specific to certain peptides. While this
provides a life-supporting protection for the brain, it
represents an insuperable obstacle for most drugs.
Therefore, a large number of drugs for the treatment
of CNS diseases or brain tumours cannot cross the
blood brain barrier in pharmacologically effective
amounts (1-3).
One possibility to increase the amount of drug in the
brain is to increase the dose applied; however, this often
leads to strong peripheral side effects, especially in case
of cytostatic drugs. Another very efficient possibility to
overcome this problem is the use of biodegradable
polymeric nanoparticles as transport systems of
drugs and genes. Nanoparticles are solid colloidal
particles, ranging in size from 1 to 1,000 nm (usually
200-300 nm), and are a rather useful ‘drug delivery
system’ to target drugs to the brain. Drugs
are attached to the nanoparticles by adsorption,
incorporation or encapsulation. Besides nanoparticles,
different techniques for the transport of drugs across
the BBB can be used.
The first drug to be successfully delivered to the brain of
animals was the hexapeptide, dalargin. Furthermore,
other drugs – for example, kytorphin, loperamide,
tubocurarine and the cytostatic doxorubicin – have also
been transported across the BBB. It has been shown that
only nanoparticles coated with polysorbate 80, or
stabilised with polysorbate 85 are able to cross the BBB.
The evidence is consistently mounting that important
diseases of the brain can be treated by a combined
nanoparticle/drug approach, and – in particular – brain
tumour treatment has been achieved in animal models.
A variety of experiments have been carried out to
date using various animal species, different
behavioural paradigms and disease models,
all of which substantiate the value of
nanoparticles as a novel drug delivery
method (4,5).
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GENE DELIVERY USING
PBCA-NANOPARTICLES
Plasmid Delivery
Brain tumours belong to the most aggressive group of
cancers in humans, with 180,000 new cases (primary and
secondary brain tumours) reported in the US per year. In
particular, glioblastomas exhibit a high mortality rate
because, in most cases, they are associated with the
development of relapses after chemotherapy and
radiotherapy. Features responsible for the aggressiveness of
glioblastomas include invasion into distant brain areas,
diffuse growth properties and extensive angiogenesis.
Neovascularisation, as a main property of glioblastomas,
indicates the possibility for anti-cancer treatment with
chemotherapeutic agents. However, insufficient response
to anti-cancer drugs is caused by the inaccessibility of the
tumour tissue due to the blood brain barrier, as well as the
loss of function of tumour suppressor genes (p53) (6,7).
The development of strategies for gene delivery across
the blood brain barrier is of enormous importance,
offering the possibility to use highly active
chemotherapeutics, together with wild-type tumour
suppressor genes controlled by a strong heterologous
promoter. At NanoDel Technologies, we have analysed
the transport of a reporter gene across the BBB into the
brain and tumour tissue. The plasmid DNA was
adsorbed onto the surface of polybutylcyanoacrylate
nanoparticles (PBCA NPs). After systemic application of
loaded NPs into rats with brain tumours, expression of
the reporter gene was observed in the tumour tissue.
Rats with implanted glioblastoma treated with βgalactosidase reporter bound to polysorbate-coated NPs
showed a significant expression of the reporter in brain
endothelial cells and liver tissue 24 hours after
application. The expression was still apparent 48 hours
later in the brain endothelial cells, in addition to strong
expression in glial cells and neurons. Expression could
also be visualised in tissue from the liver, spleen, kidneys
and stomach (8,9). The strongest expression of the βgalactosidase reporter gene was shown in brain tumours
24, 48 and 72 hours after systemic injection (see Figure
1a), with nearly all tumour cells exhibiting precipitates
from the reporter. No expression was apparent in the
heart and muscle tissue.
In summary, the study showed the distribution of a
reporter gene product in the organs of tumour-bearing
rats after binding of the gene to nanoparticles and
systemic application. Time-dependent transport of the
gene was shown across the endothelial cells and glial cells
Innovations in Pharmaceutical Technology
into the neurons of the animals;
however, the strongest expression
was shown in the experimental
tumours in the brains of the
animals. The injection of naked
control DNA did not render any
expression at all (see Figure 1c).
Antisense
Oligonucleotide Delivery
The immune system has the
ability to eliminate small tumour
cells in the human body at an early
stage of tumour growth; however,
because particularly aggressive
tumours, such as glioblastomas,
escape immune surveillance,
immunotherapeutic treatments
have been largely unsuccessful.
This immunodeficiency of
glioblastoma cells is caused by
the presence of transforminggrowth-factor TGF-β2, which
renders the cells unresponsive to
immune attacks.
Figure 1: Transport of β-Gal plasmid
and antisense oligonucleotide against
TGF-β2 into the brain of rats
Nanoparticle-mediated transport of HPV18-URRβ-Gal plasmid to F 98 glioma cells
Nanoparticle-mediated transport
of FITC-labelled antisense oligonucleotides
to F 98 glioma cells
Control: injection of pure HPV
18-URR- β -Gal plasmid
Transforming-growth-factor
produced by glioblastoma cells is
thought to be a major factor in
causing this immunosuppression.
TGF-β2 is capable of inhibiting
T cell and B cell activation and
proliferation, it suppresses the
activity of natural killer (NK)
cells, reduces the production of
cytokines such as IL-2, IL-6, ILControl: injection of pure FITC-labelled antisense
10 and IFN-γ, and suppresses the
oligonucleotides.
expression of human leukocyte
antigen (HLA)-DR on glioma
cells (10,11). Cell culture
examination has demonstrated
that expression of TGF-β2 by
glioblastoma cells could be
reduced by the use of antisenseoligonucleotides (AONs) against
m-RNA of TGF-β2 (12). AONs
are short oligonucleotides of
DNA which selectively bind to complementary m-RNA
inside the cytoplasm and, in this way, block the
production of specified proteins, such TGF-β2.
However, there is currently no method that reliably
and safely delivers genes and AONs across the blood
brain barrier.
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In conclusion, our research clearly
shows that nanoparticles are a useful
and universal method to deliver small
molecules, peptides and DNA into the
brain. Thus, polybutylcyanoacrylate
nanoparticles open up new possibilities
for the treatment of disorders of the brain
that were previously inconceivable –
such as gene therapy for brain cancer.
Nanotechnology may thus hold great
promise in this area of medicine.
Fluorescently labelled antisense oligonucleotides (labelled
with fluorescein isothiocyanate, FITC) against TGF-β2
were loaded onto PBCA nanoparticles and injected
intraperitoneally into glioma-bearing male Fisher rats. The
injection of pure FITC-labelled antisense oligonucleotides
served as control. Six hours after the single injection, all
animals were sacrificed, plasma probes were taken and
brains were examined histologically after immunehistochemical staining with fluorescein. Compared with
the control group treated with pure FITC-AON (see
Figure 1d), the nanoparticle preparation of AON (see
Figure 1b) improved diffusion across the blood-brain
barrier and uptake into the tumour – as well as into
normal cerebral tissue (13).
deliver small molecules, peptides and DNA into the
brain. Thus, polybutylcyanoacrylate nanoparticles open
up new possibilities for the treatment of disorders of the
brain that were previously inconceivable – such as gene
therapy for brain cancer. Nanotechnology may thus hold
great promise in this area of medicine.
The author can be contacted at [email protected]
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CONCLUSION
In conclusion, our research clearly shows that
nanoparticles are a useful and universal method to
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