See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/15128789

In Vitro Permeability of PBCA Nanoparticles

through Porcine Small Intestine

Article in Journal of Drug Targeting · February 1993

DOI: 10.3109/10611869308998761 · Source: PubMed

CITATIONS READS

40 19

4 authors, including:

Rolf Kinne Jörg Kreuter

Max Planck Institute of Molecular Physiology Goethe-Universität Frankfurt am Main
433 PUBLICATIONS 12,346 CITATIONS 338 PUBLICATIONS 17,597 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

SGLT-inhibitors as antidiabetic drugs View project

Brain delivery by nanoparticles View project

All content following this page was uploaded by Jörg Kreuter on 07 June 2016.

The user has requested enhancement of the downloaded file.

 Journal of Drug Targeting

ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20

In Vitro Permeability of PBCA Nanoparticles

through Porcine Small Intestine

Dieter Scherer, Frank C. Mooren, R. K. H. Kinne & Jörg Kreuter

To cite this article: Dieter Scherer, Frank C. Mooren, R. K. H. Kinne & Jörg Kreuter (1993) In
Vitro Permeability of PBCA Nanoparticles through Porcine Small Intestine, Journal of Drug
Targeting, 1:1, 21-27, DOI: 10.3109/10611869308998761

To link to this article: http://dx.doi.org/10.3109/10611869308998761

Published online: 28 Sep 2008.

Submit your article to this journal

Article views: 49

View related articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=idrt20

Download by: [Johann Christian Senckenberg] Date: 07 June 2016, At: 08:13
 Journal of Drug Targeting, 1993, Vol. 1, pp. 21-27 01993 Harwood Academic Publishers GmbH
Reprints available directly from the publisher Printed in the United States of America
Photocopying permitted by license only

In Vitro Permeability of PBCA Nanoparticles through

Porcine Small Intestine
DIETER SCHERER,’ FRANK C . MOOREN,’ R. K. H. KINNE’ and JORG KREUTER’*
’Znstitut fur Pharmazeutische Technologie der J . W . Goethe Universitat, W-6000 Frankfurt a. M . , Germany
2Max-Planck-lnstitut fur Systemphysiologie, W-6400 Dortmund, Germany

Peroral nanoparticle-mediated drug absorption was studied using a laser scanning confocal
microscope. Additional diffision studies in side-by-side diffusion cells with radiolabelled
Downloaded by [Johann Christian Senckenberg] at 08:13 07 June 2016

polybutylcyanoacrylate (PBCA) nanoparticles were carried out to confirm the results of this study.
Fluorescence-labelled PBCA nanoparticles were incubated in vitro in the lumen of freshly excised
intestine. Computer-aided optical sectioning of thick samples with dramatically improved
resolution and the possibility of rejecting out-of-focus noise enabled tracking of the fluorescence-
labelled PBCA nanoparticles in the intestinal tissue after incubation of the particles in freshly
excised porcine small intestine. The results of this study suggest that the nanoparticles are
adsorbed by the surface of the gut wall, creating a high concentration gradient, thereby enhancing
the absorption of drugs that may be loaded to the nanoparticles. A significant amount of particles
was found in hot (very fluorescent) spots that were assumed to be Peyer’s patches. No particles,
however, traversed the entire gut wall over a period of 2 to 4 h. These results were confirmed by
the diffusion study. No radioactivity permeated through Peyer’s-patch-free intestine within 4 h,
whereas the amount of radioactivity that was transported through intestine with Peyer’s patches
during this time was 1.1% of the total amount in the donor chamber.

KEYWORDS:polybutylcyanoacrylate nanoparticles, nanoparticle-mediated intestinal drug absorption, laser scanning confocal

microscope, Peyer’s patches

INTRODUCTION Three possible routes of peroral uptake of small

particles exist: (1) intracellular uptake, (2) inter-
The phenomenon of particle uptake from the cellular/paracellular uptake, and (3) uptake via the
gastrointestinal (GI) tract has been the subject of M-cells and Peyer’s patches in the gut (Kreuter,
many publications since its detection in 1844 by 1991). There is evidence that the route via the
E. F. G. Herbst. A number of research groups M-cells and Peyer’s patches seems to play a
have studied the fundamentals of particle absorp- significant, possibly dominant role in particle
tion in the past decades (LeFevre and Joel, 1977; uptake. If the other routes are involved, then they
LeFevre et al., 1989; Volkheimer and Schulz, probably play minor roles (Weiner, 1988; Eldridge
1968). The interest in this topic has increased very et al., 1990).
much over recent years due to the tendency to The total amount of particle uptake generally
use particulate carrier systems for oral drug seems to be very low. Weiner (1988) showed that
administration (Damge et al., 1990; Kreuter, 1991) the amount of uptake of several macromolecules
or for oral vaccination (Eldridge et al., 1990). amounted to only a few tenths of 1%.
The route and the extent of absorption of small In the present study, laser scanning confocal
particles is still causing controversy. One reason microscopy was used to provide direct informa-
for this is that, on one hand, the relatively small tion on the transport of fluorescence-labelled
molecule polyethylene glycol 4000 is commonly nanoparticles by the gut wall. Laser scanning
used as a nonabsorbable GI marker (Donovan confocal microscopy is a technique in which the
et al., 1990), whereas, on the other hand, specimen is illuminated and scanned point-by-
relatively large particles have been detected in point with a finely focused laser beam (Rojana-
blood capillaries after peroral administration. sakul et al., 1990). Since the viewing of the
illuminated point is performed with a spatially
* Corresponding author restricted optical system, the out-of-focus noise is

21
 22 D. SCHERER et al.

dramatically reduced and consequently, the labelled particles were prepared the same way as
resolution and the contrast are improved drama- the labelled particles.
tically in comparison to conventional systems.
Laser scanning confocal microscopy is the first
Particle Size
microscopy system that allows computer-aided
optical sectioning. For this reason, it is possible to The size of the fluorescent PBCA nanoparticles
look directly inside the specimen and localize was determined by photon correlation spectro-
exactly the position of the particles. Therefore, it scopy (PCS) (Brookhaven Inst., BI-90, Ronkon-
is possible to visualize sharp fluorescent images at koma, New York, USA).
various levels in the gut wall.
In addition to the experiments with the laser
In Vitro Perfusion Studies
scanning confocal microscope, porcine small
intestine with and without Peyer’s patches was Porcine small intestine from freshly sacrificed
placed between the chambers of side-by-side animals was obtained from the abattoir and stored
Downloaded by [Johann Christian Senckenberg] at 08:13 07 June 2016

diffusion cells and the transport of radioactivity in Ringer solution at 4°C to keep the tissue alive
through the intestine was determined using 14C- during transportation to the laboratory. The
labelled polybutylcyanoacrylate (PBCA) nano- lumen of the intestine was rinsed with 0.9% NaCl
particles. solution to remove food particles and was then
filled with a 0.05% solution of FITC in Ringer
buffer or a freshly prepared suspension of 1%
MATERIALS AND METHODS nanoparticles in Ringer buffer. Both ends of the
piece of porcine small intestine were closed by
Preparation of Nanoparticles
tying tightly around the ends with thread.
Fluorescent polybutylcyanoacrylate (PBCA) nano- The intestine was placed into a tissue bath
particles were prepared by emulsion polymeriza- immediately after preparation, and the bath was
tion, according to a method described by Kreuter gassed with carbogen (5% C02: 95% 02)to
(1983). Butylcyanoacrylate monomer (500 p; provide oxygen and to maintain circulation of the
Sichel-Werke Hannover, Germany) was added tissue bath. All experiments were carried out at
drop by drop to a solution of 50ml 0.01~-HCl 37°C. After 2 or 4h, the intestine sections were
containing 500 mg fluorescein isothiocyanate taken out of the tissue bath and the nanoparticle
(F1TC)-labelled dextran 70,000 (Sigma, St. Louis, suspension was removed. The sample was
USA). The solution was stirred with a magnetic washed with 0.9% NaCl solution in order to
stirrer for 4h. The resulting suspension was remove loosely adhering particles. The intestine
buffered with 0.1M-NaOH to pH6. The nano- was cut open and immediately shock-frozen in
particle suspension was purified prior to use by liquid air. The samples were stored frozen until
ultracentrifugation to remove unincorporated examination by laser scanning confocal micro-
FITC-dextran. The centrifugation was performed scopy was performed.
at 50,OOOg for 60min and the particles were
resuspended in distilled water. This procedure
Laser Scanning Confocal Microscope
was repeated twice.
Radiolabelled PBCA nanoparticles were obtained Measurements were performed with a BioRad
from Amersham (UK). The preparation was MRC-600 (Watford, UK) laser scanning confocal
performed by the method described above except microscope mounted on a Nikon microscope
that 14C-labelled butylcyanoacrylate was used (Osaka, Japan) and equipped with a 25mW
as the monomer, and dextran 70,000 (Sigma, argon-ion laser (excitation wavelengths 488 and
Deisenhofen, Germany) and Poloxamer 188 514nm). A Zeiss Fluor (Oberkochem, Germany)
(Erbsloh, Diisseldorf, Germany) were employed 25xwater (N.A. 1) objective lens was used. The
as stabilizers. principle of confocal microscopy is illustrated in
The radiolabelled particles were diluted with Fig. 1. The improvement of lateral and axial
non-labelled particles to prepare a stock solution resolution with laser scanning confocal micro-
for the radioactive diffusion study. These non- scopy is of special importance for measurements
 PBCA NANOPARTICLE TRANSPORT
Photomultiplier
Detecting aperture
Dichroic mirror
Downloaded by [Johann Christian Senckenberg] at 08:13 07 June 2016
Objective lens
- Ringer buffer solution and 0.5 ml stock solution of
i\/ i
Specimen \\ II
---------I---------
FIGURE 1. Schematic diagram of a laser scanning confocal
microscope. The excitation beam is reflected by the dichroic
mirror and focused on the region of interest of the specimen
by the objective lens. The emittent light passes through the
dichroic mirror and is registered by the photomultiplier. The
two aperture planes in confocal positions with respect to the
illuminated volume element of the specimen are of major
importance. They enable the exact illumination of a single
point and guarantee that only light emitted by this single point
reaches the detector. As a consequence, light from regions
above and below the focal plane is rejected resulting in
an optimal resolution. No out-of-focus blur impairs image
formation. A computer image is then created either by
scanning the excitation beam above the specimen or, alterna-
tively, the specimen can be moved together with the
microscope table along a fixed beam. In the case of the MRC-
600 microscope two scan mirrors move the laser beam across
the specimen. The pictures can be displayed in three sets of
false colours. They also can be processed and analysed by
several system-immanent program files, e.g., to measure
length, intensity, etc. Dashed lines represent regions out of
focus; solid lines represent regions in focus.
of thick specimens or for measurements below
the tissue surface. The maximum depth of pene-
tration is around l m m (Shuman et al., 1989).
Microscope Measurements
The gut wall was mounted on the microscope
table and covered with Ringer solution. Two
kinds of optical sectionings were performed.
First, horizontal sections were made to get an
impression of distribution as well as of the depths
of penetration of the fluorescent particles in the
gut wall. When the microscope was operated in
this mode, the optical plane was lowered step-
wise into the specimen. A horizontal optical
23
section was scanned at every 10pm of optical
penetration.
Second, vertical sections through a certain
region of interest were made. In this case, the
laser beam scanned in a single line across the
specimen while a motor lowered the microscope
table in a stepwise manner.
Radioactive Diffusion Studies
Higuchi-type side-by-side diffusion cells were
used for the radioactive diffusion studies. Porcine
small intestine from freshly sacrificed animals
with and without Peyer’s patches was placed
between the donor and acceptor chambers of the
diffusion cells. The diffusion cells were filled with
the radiolabelled PBCA nanoparticles was added
to the donor chamber.
The temperature was kept at 3 7 T , the dif-
fusion cells were stirred vigorously and gassed
with carbogen (5% C02: 95% 02).
Samples were taken from the acceptor chamber
after 30, 60, 90, 120, 150, 180, 210 and 240min and
replaced by fresh buffer. The radioactivity of the
samples was determined by liquid scintillation
counting.
RESULTS AND DISCUSSION
The size of the fluorescence-labelled particles
determined by PCS was 211f8 nm.
PBCA nanoparticles were labelled with FITC-
dextran because it is much more stable than the
adsorption or incorporation of low-molecular-
weight dyes. These low-molecular-weight dyes
may desorb or diffuse out of the intact particles
very rapidly and, consequently, a discrimination
between particle-bound and released dye is
difficult or impossible. FITC-dextran, on the other
hand, has been shown to be incorporated into the
polymer matrix and bound covalently (Douglas
et al., 1985). Additionally, it has been proven to
be very resistant against degradation by the
mucus of the porcine GI tract. After 24h of
incubation, only a slight decrease of the molecular
weight of FITC-dextran could be detected and no
release of FITC was found (Henry et al., 1991).
Due to this effective incorporation into the
polymer matrix of the particles, it will only be
released upon complete degradation of the
particles.
 24 D. SCHERER et al.
Downloaded by [Johann Christian Senckenberg] at 08:13 07 June 2016

FIGURE 2. Horizontal section of porcine gut wall, after 4h of incubation with FITC. The
section displayed the depth of 30pm (see Colour Plate I at the back of this publication).

FIGURE 3. Vertical section of porcine gut wall after 4h of incubation with FITC (see Colour
Plate I1 at the back of this publication).
 PBCA NANOPARTICLE TRANSPORT 25
Downloaded by [Johann Christian Senckenberg] at 08:13 07 June 2016

FIGURE 4. Horizontal section of porcine gut wall, after 4 h of incubation with FITC-dextran-
labelled PBCA nanoparticles. The section displayed the depth of 30 pm (see Colour Plate 111
at the back of this publication).

FIGURE 5. Vertical section of porcine gut wall, after 4 h of incubation with FITC-dextran-
labelled PBCA nanoparticles (see Colour Plate IV at the back of this publication).
 26 D. SCHERER et al.
Pigs have been widely used in the study of
resorption and digestion because of similarities of
their body weight with that of humans, but
especially because sections of the porcine GI tract
anatomically resemble those of humans (Hossain
et al., 1990). For this reason GI tissues of these
animals were used for this study.
Horizontal sections of tissue incubated 4 h with
FITC showed a more or less uniform distribution
of fluorescence (Fig. 2). Vertical sections showed
the highest accumulation of fluorescence at a
depth of about 50pm. The maximum depth of
penetration was approximately 125pm (Fig. 3).
The distribution of fluorescence in horizontal
Downloaded by [Johann Christian Senckenberg] at 08:13 07 June 2016
sections of intestinal tissue incubated 4 h with
FITC-dextran-labelled nanoparticles, on the other
hand, was very variable. There was a high
accumulation of fluorescence in some areas,
whereas in other nearby areas only small amounts
of fluorescence were detectable (Fig. 4). Vertical
sections (Fig. 5) showed a thin layer of fluores-
cence with a maximum depth of penetration of
the nanoparticles of approximately 60 pm. This
shows that the penetration of nanoparticles into
the gut wall over a period of 4 h is limited to the
first two or three cell layers of the gut wall.
Complete penetration of particles through the
entire gut wall was never observed during this
time period.
As mentioned above, the distribution of fluor-
escence with nanoparticles was very variable,
with a high accumulation in some defined areas
in the gut wall and with minor amounts of
fluorescence in other parts. It was assumed that
the areas with high accumulation of nanoparticles
represent lymphatic follicles or Peyer’s patches.
These results were confirmed by the results of
the transport of radioactivity through Peyer’s
patches and Peyer’s-patch-free small intestine. No
permeation of radioactivity through patch-free
small intestine was observable within the 4 h time
frame of the study (Fig. 6 ) . The amount of
radioactivity that permeated through the Peyer’s-
patch-containing sections of the intestine within
4 h was about 1.1% of the applied dose. Part of
this radioactivity may result from degradation
products and not from intact particles due to the
high amount of esterase enzyme in Peyer’s
patches (Wolf and Bye, 1984), which can degrade
polyalkylcyanoacrylate nanoparticles.
These results suggest that adhesion of nano-
- 0
0 Peyer’s
Peyer’s patches
patches
:mm Patch-free mucosa
1.5 -
t
--
1.0
-
0’
0.5
0 - - -- - -- I
0 30 60 90 120 150 180 210 240min
FIGURE 6. Permeation of radioactivity in percent of the total
dose in the donor-chamber through Peyer’s patches (0) and
Peyer’s-patch-free (W) tissue from porcine small intestine.
particles to the gut wall and, as a consequence,
the creation of a high drug concentration gradient
as well as transport through Peyer’s patches,
were possibly responsible for the enhancement of
the GI drug absorption by nanoparticles that has
been observed previously (Maincent et al., 1986).
Our own recent unpublished studies in compar-
able in vitro experiments with porcine small
intestine have shown that although no nano-
particle transport through the intestinal wall was
observed, the transport of a number of drugs
bound to nanoparticles, nevertheless, was
enhanced in comparison to a drug solution.
However, the results in this study like all in vitro
studies have to be interpreted with certain caution,
because potential transport mechanisms may not
be functional in excised tissue.
The preferential accumulation of nanoparticles
in Peyer’s patches may be very useful for the oral
administration of vaccines (Eldridge et al., 1990)
and possibly also for immunomodulating drugs.
ACKNOWLEDGMENTS
The support of this work by Lohmann Therapie-
Systeme, Neuwied, Germany, is gratefully acknow-
ledged.
(Received April 14, 1992)
(Accepted May 12, 1992)
 PBCA NANOPARTICLE TRANSPORT
References
Damge, C., Michel, C., Aprahamian, M., and Couvreur, P.
(1990). Nanocapsules as carriers for oral peptide delivery. 1.
Controlled Re/., 13, 233-239.
Donovan, M.D., Flynn, G.L., and Amidon, G.L. (1990).
Absorption of polyethylene glycols 600 through 2000: The
molecular weight dependence of gastrointestinal and nasal
absorption. Pharm. Res., 7, 863-868.
Eldridge, J.H., Hammond, C.H., Meulbroek, J.A., Staas, J.K.,
Gilley, R.M., and Tice, T.R. (1990). Controlled vaccine
release in the gut-associated lymphoid tissues. I. Orally
administered biodegradable microspheres target the Peyer’s
patches. 1. Controlled Re/., 11, 205-214.
Henry, B.T., Cheema, M.S., and Davis, S.S. (1991). Gel
permeation chromatography used to determine the stability
of FITC-dextrans in human saliva and porcine small
intestine mucus. Znt. 1. Phurm., 73, 81-88.
Downloaded by [Johann Christian Senckenberg] at 08:13 07 June 2016
Hossain, M., Abramowitz, W., Watrous, B.I., Szpunar, G.J.,
and Ayres, J. W. (1990). Gastrointestinal transit of nondisin-
tegrating, nonerodible oral dosage forms in pigs. Pharm.
Res., 7, 1163-1166.
Kreuter, J. (1983). Evaluation of nanoparticles as drug-delivery
systems. I. Preparation methods. Pharm. Acta. Helv., 58,
196209.
Kreuter, J. (1991). Peroral administration of nanoparticles.
View publication stats
27
Adv. Drug. Del. Rev., 7, 71-86.
LeFevre, M.E. and Joel, D.D. (1977). Intestinal absorption of
particulate matter. Life Sci., 21, 1403-1408.
LeFevre, M.E., Boccio, A.M., and Joel, D.D. (1989). Intestinal
uptake of fluorescence microspheres in young and aged
mice. Proc. Soc. Exp. Biol. Med., 190, 23-27.
Maincent, P., Le Verge, R., Sado, P., Couvreur, P., and
Devissaguet, J.P. (1986). Disposition kinetics and oral
bioavailability of Vincamine loaded polyalkylcyanoacrylate
nanoparticles. 1. Pharm. Sci., 75, 955-958.
Rojanasakul, Y., Paddock, S.W., and Robinson, J.R. (1990).
Confocal laser scanning microscopic examination of trans-
port pathways and barriers of some peptides across the
cornea. Int. 1. Pharm., 61, 163-172.
Shuman, H., Murray, J.M., and DiLullo, C. (1989). Confocal
microscopy: An overview. BioTechniques, 7, 154-163.
Volkheimer, G. and Schulz, F.H. (1968). The phenomenon of
persorption. Digestion, 1, 213-216.
Weiner, M.L. (1988). Intestinal transport of some macromole-
cules in food. Fd. Chem. Toxic., 26, 867-880.
Wolf, J.L. and Bye, W.A. (1984). The membraneous epithelial
(M) cell and the mucosal immune system. Ann. Rev. Med.,
35, 95-112.
Zimmer, A,, Kreuter, J., and Robinson, J.R. (1992). Studies on
the transport pathway of PBCA nanoparticles in ocular
tissue. 1. Microencapsulation (in press).