PEPTIDE-DERIVATIZED BIODEGRADABLE NANOPARTICLES ABLE TO CROSS THE BLOOD BRAIN
BARRIER
G. Tosi, L. Costantino, F. Gandolfi, B. Ruozi, E. Leo, M.A. Vandelli, F. Forni.
Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia
E-mail: tosi.giovanni@unimore.it
Key words: Blood Brain Barrier, Nanoparticle, Peptide, Brain Perfusion, Fluorescent studies
INTRODUCTION
Injectable nanoparticulate drug carriers (Np) able to
cross the blood brain barrier (BBB) have important
potential applications for the treatment of diseases
affecting the central nervous system (CNS). To date,
direct evidence of BBB crossing was given only by
hydrophobic
Np,
mainly
constituted
by
polyalkylcyanoacrylate [1,2] surface-modified by
physical adsorption of a hydrophilic polymer, for
example the surfactant polysorbate 80, or by Np made
of an amphiphilic copolymer in which the hydrophobic
block itself is able to form a solid phase, while the
hydrophilic part remains on the surface, like the block
copolymer
polyethylenglycol
and
nhexadecylcyanoacrylate (PEG-PHDCA). With the aim
to create a system able to address Np to the CNS, we
developed new conjugates formed by a biodegradable
copolymer, poly(D,L-lactide-co-glycolide) (PLGA) or
a fluorescein/rhodamine-PLGA conjugate [3]. linked
with short peptidic sequences based on the structure of
the opioid peptides; these modified copolymers seem to
be a valuable starting material for the preparation of Np
that possess the peptidic moieties on their surface.
EXPERIMENTAL METHODS
Nanoparticles were manufactured according to the
nanoprecipitation procedure [4]. The new polymer
PLGA RG503H conjugated with peptides and PLGA
RG503H derivatized with fluorescein were dissolved in
acetone. The organic phase was added dropwise into
deionised water containing Pluronic F68. After stirring
at room temperature for 10 min, the organic solvent
was removed at 30° C under reduced pressure. The
final volume of the suspension was adjusted with
deionised water; Np were then purified by gel-filtration
chromatography (Sepharose CL 4B gel, column 50 x 2
cm), using water as mobile phase and freeze–dried
without any cryoprotector. A scanning electron
microscope (SEM) (XL-40 Philips, Eindhoven, The
Netherlands) was used to evaluate the Np morphology.
The samples were coated under argon atmosphere with
a 10-nm gold palladium thickness (Emitech K550
Supper Coated). The Np present an homogeneous
distribution size and integrity of their surface (Fig. 1).
To determinate the position that hydrophilic groups
modifying the polymer assumed during Np formation,
X-ray photoelectron spectroscopy (ESCA) analysis was
performed on an analysis system 04-153 X-ray source
(PHI, Uvalca-PHI, Tokyo, Japan) and an hemispherical
electron analyser EA11 (Leybold Optics, Germany),
using MgK 1,2 radiations The spectra were recorded
in FAT (fixed retardation ratio) mode with 190 eV pass
energy. The pressure in the sample analysis chamber
was ca. 10−9 mbar. Data acquisition and processing
were performed with the RBD AugerScan 2. The
spectra showed the presence of N on the surface of Np,
and the signal was greater in presence of PLGApeptide (80%) and PLGA-fluorescein conjugate (20%)
(Fig. 2) than the N signal present in the spectrum
obtained using Np made of PLGA (80%) and PLGAfluorescein conjugate (20%) (Fig. 3), as expected.
RESULT AND DISCUSSION
While PLGA Np are unable to cross the BBB, for the
first time solid Np surface-modified with peptides are
shown to be able to cross the BBB.
Np were prepared with different combination of PLGA
polymer: Np A was composed by 80% PLGA RG
503H, Np B by 20% of the conjugate RG503Hfluorescein 80% PLGA RG 503 and 20% of the
conjugate RG503H-fluorescein and Np C by 80%
PLGA RG 503H-peptide and 20% of RG503Hfluorescein. The determination of the capacity of the
Np to cross the BBB was assessed by an in vivo assay
(the In-situ Rat Brain Perfusion Technique [5,6]). Each
Np sample was perfused in rats as suspension for 60
seconds, followed by a perfusion of simil-plasma fluid
(120-180 s). Rats were then sacrificed, brain removed
and submitted to conservative procedures by washing
with saline solution and then frozen with liquid N2.
Each frozen tissue was cut in all its thickness by a
cryotome into 5 m slides (about 40 slides each brain).
To evidence the presence of the fluorescence
associated to Np, the slides were treated with DAPI (4'6-diamidino-2-phenylindole), which is known to form
fluorescent complexes with natural double-stranded
DNA [7]. After treatment of the slides with DAPI, the
samples were observed using a fluorescence
microscope with dual excitation band DAPI and
fluorescein isothiocyanate (FITC) using an emission
filter set for fluorescence imaging (40-100x
magnification) and with a confocal microscope (Leika
DM IRE 2). Fluorescein and tetramethylrhodamine
conjugates, giving green and red fluorescent spots,
respectively, were considered the visible markers of
nanoparticles (Fig. 4).
Then, in order to allow a more precise location of the
Np inside the nervous parenchyma, several studies on
confocal microscopy have been performed. These
details cannot be achieved from fluorescent
microscopy studies, which give informations only in a
bidimensional (2D) modality. The confocal images,
owing to the 3D possibility of investigation,
demonstrate the positions of Np (green fluorescence
due to fluorescein) relating to the nucleus (blue
fluorescence due to DAPI) of nervous cells. The Np
(Fig. 6) are in a close contact with nuclear structures; in
some cases, they are at the same level of nucleus, and
in other cases they are located into the cytoplasm.
Thus, in order to check the capacity of the Np
composed of the copolymer PLGA RG503H
derivatized with peptide (80%) and labelled with 20%
PLGA-tetramethylrhodamine to avoid opsonization,
these were injected into the femoral vein and after 60
min the presence of Np in the CNS was assessed. The
image (Fig. 5) allow to recognize red spots, due to
tetramethylrhodamine, that evidence the presence of
Np in brain parenchyma. Even if some of the particles
were found in the liver, it is very important to underline
that some Np were found into the CNS parenchima.
This represents a further confirmation of the important
role of the modification of the Np with the peptidic
sequences here considered in their ability to cross the
BBB and confirm also the results previously obtained
with brain perfusion experiments.
7.
Z. Xu, D.S. Pilch, A.R. Srinivasan, W.K. Olson, N.E.
Geacintov, K.J. Breslauer, Bioorg. Med. Chem. 5, 1137
(1997)
Figure 1. Np SEM image
Figure 2-3. ESCA studies on un-modified Np (2) and modified Np (3).
Figure 4-5. Fluorescence studies on cerebral parenchyma, Np
localization consequence to a crossing of BBB (fig. 4). Rhodamine
derivatized Np: fluorescent microscopy image in cerebral tissue (fig.5)
CONCLUSION
A new polymeric conjugate was obtained with the aim
of cerebral targeting; the methods used for the
detection of Np aggregates, confocal and fluorescent
microscopy, suit perfectly to our proposal, confirming
that the new peptides here described can represent a
useful way to target CNS by Np.
ACKNOLEDGMENT
Authors wish to thank Dott. A. Tombesi and Dott. C.
Restani (CIGS, Univ. Modena and R.E.) for their
technical support of confocal microscopical analysis.
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Fig.4
Cerebral
Parenchyma
Fig.5
Figure 6. Confocal study performed in a single plane in z thickness on
cerebral parenchyma Images are referred to the intensity per cent of
green and blue coloration (respectively from fluorescent Np and
DAPI-double strand DNA complexes) The spots included into the
yellow ellipsis in figure 3C and 3D are considered as the points of
interaction between fluorescent Np and cells.