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Effects Of Chitosan Nanoparticles On Oral Absorption Of

Unwanted Toxins Biology Essay
Despite the advances in biotechnology and allied fields, successful oral administration of protein/peptide
drugs has remained to be an elusive goal for the researchers around the globe. Orally administered protein
drugs encounter several difficulties such as rapid pre-systemic degradation and poor absorption in the
gastrointestinal (GI) tract [1]. Therefore, a delivery system is needed to improve the oral bioavailability of such
drugs. An ideal delivery system for oral administration of protein drugs should provide the intact drug to the
systemic circulation and reversibly increase the permeability of the mucosal epithelium to enhance the absorption
of protein drugs [2]. Additionally, this delivery system should not show any adverse systemic effects or irreversibly
damage the intestinal epithelium.

In a recent study, we reported a pH-responsive nanoparticle (NP) system shelled with chitosan (CS) for the oral
delivery of insulin via the paracellular pathway [3, 4]. CS, a cationic polysaccharide, can adhere to the epithelial
surfaces to impart transient opening of the tight junctions between contiguous cells [5]. The results obtained in a
diabetic rat model indicated that the prepared NPs could effectively adhere to the mucosal surface, increase the
intestinal absorption of insulin and produce a slower, but prolonged hypoglycemic effect. Additionally, the in vivo
toxicity study indicated that the test NPs were safe even at a dose 18 times higher than that used in the
pharmacodynamics/ pharmacokinetics (PD/PK) study [4].

CS is a natural-origin polymer, which has been widely used as a weight-loss product and been developed as a
safe excipient in drug formulations [6]. Additionally, CS, an excellent mucoadhesive agent, has been widely used
as a paracellular permeability enhancer for improving the intestinal absorption of hydrophilic macromolecules [6,
7]. CS has been proven to be safe and biocompatible [8]. However, it is often argued that the paracellular
permeation enhancement by CS may result in absorption of unwanted toxins present in the GI tract [9]. Therefore,
the purpose of this study was to evaluate the effects of test NPs on the oral absorption of unwanted toxins present
in intestinal lumen.

Lipopolysaccharide (LPS, also known as endotoxin) is a component of the outer membrane of gram-negative
bacteria. If delivered to the systemic circulation, LPS can trigger a systemic inflammatory response that may
progress to septic shock and sometimes death in vivo [10]. Although disintegrating enteric bacteria continuously
release LPS in the intestinal lumen, the intact intestinal epithelium constitutes a barrier that prevents significant
absorption of this amphiphilic molecule [11]. Hence, the bacterial LPS was selected as a model toxin in this study.

The biodistribution of the orally administered LPS was studied in a rat model using the single-photon emission
computed tomography (SPECT)/computed tomography (CT). To understand how the intestinal epithelium
prevents the in vivo absorption of LPS, we investigated the absorption of fluorescent LPS in rats using the
confocal laser scanning microscopy (CLSM). Then, the effects of test NPs on the enteral absorption of LPS were
investigated using SPECT/CT and CLSM. Finally, the adverse effects of orally administered LPS and effects of
test NPs on them were investigated in an in vivo toxicity study.

2. Materials and Methods

2.1. Preparation and characterization of test NPs

CS (MW 80 kDa) with a degree of deacetylation of approximately 85% was acquired from Koyo Chemical Co. Ltd.
(Japan), while γ-PGA (MW 60 kDa) was purchased from Vedan Co. Ltd. (Taichung, Taiwan). A sample of 100 mg
of insulin (from bovine pancreas, 27.4 IU/mg, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10 mL of 0.01N
HCl and this solution was neutralized with 0.1N NaOH [12]. The insulin solution was then diluted with deionized
(DI) water to make a stock solution of 1 mg/mL insulin. Test NPs were prepared by a simple ionic-gelation method
previously reported by our group [4, 13].

In brief, the prepared insulin stock solution (1 mL) was premixed with an aqueous γ-
PGA (2 mg/mL, 1 mL). Subsequently, TPP (1 mg) and MgSO4 (2 mg) were added
into this mixture and thoroughly stirred for 30 min. The mixed solution was then
added by flush mixing with a pipette tip into an aqueous CS solution (1.5 mg/mL, 8
mL, pH 6.0) under magnetic stirring at room temperature. Self-assembled NPs were
collected and washed three times with DI water by centrifugation at 8000 rpm for 50
min. The centrifuged NPs were redispersed in DI water and stored at 4ºC until used.
The mean particle size and zeta potential value of NPs were measured using a
Zetasizer (Nano ZS, Malvern Instruments Ltd., Worcestershire, UK) [14].

2.2. Evaluation of the micelle forming characteristics of LPS

LPS molecules are composed of a hydrophilic oligosaccharide chain of varying length and a hydrophobic,
membrane-anchoring moiety, termed lipid A []. The amphiphilic nature of LPS results in the formation of micelles in
an aqueous environment. To confirm these characteristics, different concentrations of LPS were suspended in
PBS (pH 7.4). The formation of micelles was confirmed by measuring the particle size and zeta potential of the
resulting suspensions using Zetasizer (Nano ZS, Malvern Instruments Ltd., Worcestershire, UK).

2.3. Animal studies

Animal studies were performed in compliance with the “Guide for the Care and Use of Laboratory Animals”
prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the
National Academy Press, revised in 1996.

2.3.1. Biodistribution study

The biodistribution of LPS and the insulin loaded in NPs was studied in rats using the SPECT/CT (n = 3). In this
study, LPS (Escherichia coli, Serotype 0111: B4, Sigma-Aldrich, St. Louis, MO, USA) was radiolabeled with
99mTc-pertechnetate (99mTc, emitting 140KeV photons) using a stannous chloride (SnCl2) method [15]; the
labeling efficiency of 99mTc to LPS was determined by the instant thin layer chromatography (ITLC). The insulin
was radiolabeled with 123iodine (123I, emitting 159KeV photons) using an iodogen-tube (Pierce Iodination Tubes,
Thermo Fisher Scientific, Rockford, IL, USA) method, as per the manufacturer’s instructions. The 123I-insulin was
separated from the free-form 123I using a centrifugal dialysis device (MWCO: 3 kDa, Amicon Ultra 4, Millipore,
Billerica, MA, USA); its labeling efficiency was determined by a reversed-phase HPLC system equipped with a
gamma counter [16]. The obtained 123I-insulin was then used to prepare test NPs as described above.

To study the biodistribution, rats were fed with 99mTc-LPS (?) alone or with 123I-insulin-loaded NPs (?). For
the group receiving both 99mTc-LPS and 123I-insulin-loaded NPs, test NPs were administered 1 h after the
ingestion of 99mTc-LPS, to mimic the natural conditions where LPS is evenly distributed in the GI tract. The
detailed protocol used in the image acquisition was previously described by our
group [17, 18]. In brief, animal images were acquired using a dual modality system,
NanoSPECT/CT (Bioscan Inc., USA), which is capable of detecting two kinds of
isotopes simultaneously at high resolution (~0.6 mm). Animals were kept under the
controlled temperature (37ºC) and anesthesia (1.5% isoflurane in 100% oxygen)
during imaging. Dual isotope dynamic SPECT images were acquired at 30-min
intervals up to 24 h after the oral administration of test samples. Additional CT
images were collected for anatomical references and used to investigate the details
of radiotracer distribution in rats. The co-registered dynamic scintigraphy and CT
images were displayed and analyzed using the PMOD v2.9 image analysis software
(PMOD Technologies Ltd., Zurich, Switzerland).

Areas of the whole body and GI tract were selected for the volume of interest (VOI) analysis to obtain their
biodistribution profiles. To calculate the percentage of initial dose (% ID) within each area, the corresponding VOIs
were manually drawn on the co-registered dynamic SPECT and reference CT images. The peripheral
tissue/plasma compartment was defined as the whole body excluding the GI tract and urinary bladder. The
biodistribution data were expressed as %ID using the following formula:

(total counts in the target organ)  100 %

ingested counts

% ID/organ =

2.3.2. Ultrastructural examination of the in vivo tight junction opening activity of CS-NPs

The in vivo tight junction opening activity of test NPs was studied using transmission electron microscopy (TEM).
For this, CS was labeled with quantum dots (QD) as per a previously reported method [19]. Briefly, carboxyl QDs
(40 µL, 0.6nM, Qdot® ITK™, Invitrogen, USA) were activated in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC, 60 µL, 50mM) and N-hydroxysuccinimide (NHS, 30 µL, 25mM) for 15 min under gentle
stirring. The resulting NHS-activated QDs were covalently linked to the primary amines on CS at pH 6.0. The
reaction was carried out under gentle mixing for 4 h. The final QD-CS conjugate was purified by the centrifugal
spin filtration and resuspended in DI water. QD-CS-NPs were then prepared using the method described above.
The QD-CS-NPs (1 mg/mL, 1 mL) were then administered to the overnight-fasted male ICR mice (33–40 g,
National Laboratory Animal Center, Taipei, Taiwan). Animals were sacrificed 3 h later and the intestinal segments
were dissected and washed three times with isotonic saline. The dissected intestinal segments were then fixed in
4% paraformaldehyde. The fixed intestinal segments were cut into small pieces, washed with s-Collidine buffer
and treated with 2% lanthanum nitrate for 2 h at room temperature. After being rinsed in s-Collidine and then PBS,
the tissue samples were processed for TEM. Test samples were postfixed in 1% osmium tetroxide, dehydrated in
a graded series of ethanol, and embedded in the Spurr resin. Ultrathin sections were then cut with a diamond
knife and loaded onto the TEM grids. The sections were examined by a Philips CM10 electron microscope (Philips
Electron Optics B.V.) at accelerating voltage of 120 kV and micrographs were taken.

2.3.3. GI absorption of FITC-LPS and Cy3-insulin-loaded-NPs

FITC-labeled-LPS (FITC-LPS, Sigma-Aldrich, St. Louis, MO, USA) and Cy3-labeled-insulin were used to
visualize their in vivo absorption characteristics using CLSM (TCS SL, Leica,
Germany). The Cy3-insulin was synthesized as per the method described in the
literature [20]. Briefly, Cy3-NHS ester (GE Healthcare, Pittsburgh, PA, USA)
dissolved in DMSO (1 mg/mL, 1 mL) was slowly added to an aqueous solution of
bovine insulin (1% w/v in 0.01N HCl, 4 mL) and stirred overnight at 4ºC. To remove
the unconjugated Cy3, the synthesized Cy3-insulin was dialyzed in the dark against
5 L of 0.01N HCl and replaced on a daily basis until no fluorescence was detected in
the dialysis medium. The resultant Cy3-insulin was lyophilized in a freeze dryer.
Fluorescent NPs were then prepared for the subsequent in vivo CLSM studies as
per the procedure described above.

FITC-LPS (2 mg/mL, 0.5 mL) alone or in combination with a mucolytic agent (N-acetylcystein) were administered
to the overnight-fasted male Wistar rats (n = 3). To study the effects of CS-NPs on the LPS absorption, Cy3-
insulin-loaded NPs (2 mg/mL, 0.5 mL) were administered 1 h after the administration of FITC-LPS. Rats were
sacrificed 3 h later and intestinal segments were then dissected and washed three times with isotonic saline. The
isolated intestinal segments were fixed in Methanol-Carnoy’s fixative and processed for paraffin-embedding. The
paraffin-embedded sections were dewaxed, hydrated and stained with Alexa-633-labeled wheat-germ-agglutinin
and SYTOX blue (Invitrogen, ?, ?, USA) to visualize the mucus and nuclei, respectively. Finally, the stained
sections were examined under CLSM.

2.3.4. In vivo toxicity study

It is well known that LPS, a potent inflammatory agent, has many physiological and biochemical effects in vivo
[21]. Therefore, it was decided to evaluate the toxicity of the orally administered LPS in ICR mice. Animals were
randomly divided into four groups (n = 7 for each studied group). The experimental groups received an oral dose
of LPS (5 mg/kg) with or without blank NPs (10 mg/kg); the group without any treatment was used as a control.
Additionally, a group receiving intraperitoneal (IP) LPS (5 mg/kg) served as a reference for the extent of toxicity
produced by the systemic LPS. All animals were fed with normal chows and water ad libitum. Animals were
observed carefully for the onset of any signs of toxicity and monitored for changes in body weight. At the end of the
treatment period, animals were anaesthetized (tribromoethanol, IP, 240 mg/kg) and blood samples were collected
via cardiac puncture for the determination of alanine aminotransferase and aspartate aminotransferase using a
FUJI DRI-CHEM 3500s serum-chemistry analyzer.

2.4. Statistical analysis

Comparison between groups was analyzed by the one-tailed Student’s t-test (SPSS, Chicago, Ill). All data are
presented as a mean value with its standard deviation indicated (mean ± SD). Differences were considered to be
statistically significant when the p values were less than 0.05.

3. Results and discussions

The efficacy of oral delivery of peptides/proteins is often limited because of their inherent instability in the GI tract
and their low permeability across the epithelial membranes. The high molecular weight of this class of drugs
coupled with their hydrophilic nature restricts their transcellular permeation [1]. Thus, enhancement of the
paracellular permeation is a more feasible alternative for oral delivery of peptides/proteins [22]. CS is a well-
known mucoadhesive agent with the capability of transiently and reversibly opening tight junctions between
epithelial cells [7].

Formulating CS into NPs has advantages over the traditional tablet or powder formulations, as the NPs can
readily infiltrate into the mucus layer and deliver the protein drugs to the actual site of absorption (i.e., the
tight junctions between epithelial cells). CS is generally regarded as a safe and biocompatible material for
drug delivery. However, its effects on the absorption of unwanted toxin present in the
GI tract have never been investigated. It is often argued that the disruption of tight
junctions by CS may promote the intestinal absorption of such toxins, resulting in
systemic immunogenic reactions [9]. To our knowledge, this is a first such study
evaluating the effects of CS-NPs on the intestinal absorption of LPS (a model for
unwanted toxins present in the GI tract).

3.1. Characterization of the insulin-loaded NPs

The prepared NPs had a mean particle size of 253.2  4.8 nm with a positive zeta
potential of 28.2 ± 1.3 mV and their insulin LE and LC were 72.4  3.9 % and 17.9  2.1 %, respectively (n = 6
batches). The NPs were stable in the pH range of 2.0–7.0. The NPs prepared using QD-CS or FITC-CS exhibited
similar physicochemical properties as the NPs prepared with unmodified CS.

3.2. Oral absorption and biodistribution of LPS

The absorption and biodistribution of the orally administered LPS was investigated by the SPECT/CT. In the study,
99mTc was used to label LPS and its labeling efficiency was determined by the ITLC. It was found that more than
99% of the 99mTc was bound to the LPS (Fig. 1a). The SPECT/CT images of orally administered 99mTc-LPS are
shown in Fig. 1b. As shown, the radioactivity (99mTc-labeled LPS) propagated from the stomach, small intestine
and then to the large intestine with time. Overall, the 99mTc-labeled LPS appeared to be restricted within the GI
tract, with no quantifiable presence in the plasma and peripheral tissues. These results confirmed the previously
reported findings that the orally administered LPS was not able to be absorbed into the systemic circulation in
rabbits [23].

It is known that the epithelium lining on the GI tract provides a regulated, selectively permeable barrier between
the external environment (the intestinal lumen) and the systemic circulation [24]; it transports nutrients, ions, and
fluid tans cellular ly, but prevents the entry of toxins, antigens, and microorganisms. The paracellular route is
restricted by the tight junctions at the apical poles of enterocytes that limit the passage of macromolecules. In
general, the permeability of the intestinal epithelium depends on the regulation of intercellular tight junctions [22].
Therefore, it is important to study the effects of tight junction opening activity of CS on the transport of antigens
(e.g., LPS) present in the GI tract.

3.3. Effects of CS-NPs on the absorption and biodistribution of LPS and insulin

Effects of CS-NPs on the biodistribution of 99mTc-LPS and the loaded drug (123I-insulin) were studied
simultaneously using the dual isotope dynamic SPECT/CT in a rat model following oral administration. To mimic
the physiological conditions, 99mTc-LPS was ingested 1 h prior to the administration of 123I-insulin-loaded NPs.
As shown in Fig. 2a, with time progressing, 99mTc-LPS propagated from the stomach, small intestine and then to
the large intestine; the radioactivity associated with LPS was mainly limited to the intestinal lumen throughout the
entire course of the study. On the other hand, 123I-insulin started to appear in the urinary bladder at 3 h post
administration. These results suggest that the insulin loaded in NPs can traverse the intestinal epithelium and
enter the systemic circulation, whereas LPS was unable to do so, hence retaining within the intestinal lumen.

The biodistribution data in Fig. 2a was further processed for the quantification of LPS and insulin entering
into the plasma/peripheral tissues. For this, a 3D VOI was constructed; the plasma/peripheral tissue
compartment was defined as the whole body (blue contour) excluding the GI tract and urinary bladder
(green contour). Dynamic 99mTc-LPS SPECT/CT images overlay with VOI contours
in the coronal, sagittal, and transverse views are presented in Fig. 2b. The contour
marked with blue line was identified as the whole body, the contour marked with
green line was identified as the GI tract and urinary bladder. As previous mentioned,
the PP compartment was defined as volume between blue and green contours. The
radioactivity counts of 99mTc-LPS and 123I-insulin within the PP compartment were
calculated and normalized to each initial ingested dose (%ID/compartment) for
comparison. As shown in Fig. 2b (iv), the distribution of 123I-insulin in the PP
compartment increased slowly and reached to its maximal value (~8%ID) by 6 h post
administration. On the other hand, no measurable 99mTc-LPS could be observed in
the PP compartment, indicating that the paracellular permeation effect of test NPs was specific for the loaded
insulin. To determine the specific reasons for inability of LPS to pass through the opened paracellular route, we
evaluated the physicochemical properties of the LPS in aqueous environment.

3.4. Micelle forming characteristics of LPS

It has been reported that the amphiphilic character of LPS results in the formation of micelles in an aqueous
environment above their critical micellar concentration (CMC) [25]. The reported CMC values for LPS vary from 10
nM to 1.6 µM depending on the source of LPS [26]. Based on this concentrations and the fact that the LPS
aggregates are usually highly stable, it was suggested that the aggregate forms of LPS should predominate in the
concentration range relevant for biological responses [25]. To evaluate the micelle forming characteristics of LPS,
we measured the size and zeta potential of the aqueous suspensions of LPS at different concentrations.

Figure 3 shows the effects of the concentration on the average size and particle size distribution of the LPS
micelles. With an increase in the concentration, the average size of LPS micelles increased significantly. This
could be attributed to the aggregation of multiple micelles at the higher concentrations, which was also evident
from the particle size distribution profiles of these micelles. As shown, the size distribution for LPS at lower
concentration (0.2 mg/mL) was narrow indicating that the LPS predominantly exist as dispersed micelles at lower
concentration. However, at higher concentrations of LPS (2 mg/mL and 10 mg/mL), the particle size distribution
became broader indicating the formation of bigger aggregates of LPS. The zeta potential of these micelles was
anionic at all concentrations. The anionic charge on the LPS micelles could be a result of two phosphate groups
on the LPS structure (Fig. 3).

Based on these findings, we hypothesized that the anionic micelles and aggregates of LPS would be repelled by
the negatively charged mucus layer on intestinal epithelium, thus preventing them from entering the systemic
circulation. On the other hand, the test NPs with mucoadhesive CS on their surfaces may adhere and infiltrate into
the mucus. The infiltrated NPs then disintegrate near the cell surfaces due to their pH-responsive characteristics
and release the loaded insulin. The released insulin could then enter the systemic circulation due to the TJ
opening activity of CS (Figure 4).
3.5. Ultrastructural examination of TJ opening activity of CS in vivo

The ability of CS to increase the permeability of model drug compounds across Caco-2 cell monolayers has
been investigated in numerous studies. However, very few studies have focused on
the ultrastructural examination of its TJ opening activity in vivo. Figure 5a shows the
TEM micrographs of the intestinal sections from mice treated with QD-CS NPs. As
shown, the QD-CS-NPs were adhered to the microvilli and goblet cells indicating that
they were able to infiltrate the mucus layer. However, a very small fraction of QD-CS
could be observed within paracellular spaces. It has been reported that the size of
TJs opened by CS is less than 20 nm [22]; this could be the reason for poor
permeation of QD-CS in the paracellular spaces. Therefore, to visualize the TJ
opening activity of CS, we examined the movement of lanthanum (La3+) through the
opened TJs. La3+ is an electron dense element with a hydrated radius (0.4 nm),
which has been widely used a surface coating material for several tissues [27]. As shown in Figure 5b, La3+ could
penetrate the paracellular spaces, indicating that the TJ were indeed opened by the QD-CS NPs. A careful
examination by TEM revealed no detectable alterations in the morphology of the tight junctions or lateral
intercellular spaces, indicating that the TJ opening activity of CS was transient and reversible. These results
supported our hypothesis that the test NPs could infiltrate the mucus layer on the intestinal epithelium and could
release the loaded drugs near cell surface to promote its absorption.

3.6. In vivo CLSM study

Mucus is a viscoelastic gel layer that protects tissues that would otherwise be exposed to the external
environment [28]. The thickness of the intestinal mucus layer has been determined in several studies. Atuma and
co-workers reported that the mucus layer was thickest in the colon (~830 µm) and thinnest in the jejunum (~123
µm). They also reported the presence of an inner, firmly adherent mucus layer in the stomach (~80 µm), and colon
(~116 µm). In the small intestine, this layer was very thin (~20µm) or absent [29]. The protective functions of this
adherent mucus gel layer have been widely investigated [30]. In the stomach and duodenum, it provides a stable
unstirred layer supporting surface neutralization of luminal acid by mucosal bicarbonate secretion. In this way, it
maintains a pH gradient from acid in the lumen to near neutral pH at the mucosal surface [31]. In addition, the
mucus layer has been shown to act as a physical barrier to the enteric bacteria in colon and hinders their access
to the underlying epithelium [Johansson, PNAS 2008]. However, the protective effects of the mucus layer against
the LPS are still unknown.

In present study, we hypothesized that the mucus layer plays an important role in preventing the intestinal
absorption of LPS. To prove this hypothesis, the in vivo absorption of LPS was studied at a microscopic level
using the CLSM. For this, the FITC-LPS with or without a mucolytic agent (N-acetylcystein) were administered to
the overnight fasted rats. It was found that the mucus layer was intact under normal circumstances (Figure 6,
upper panel). The administration of N-acetylcystein caused the mucus layer to detach from the intestine due to the
mucolytic effects of this agent (Figure 6, lower panel). The FITC-LPS was found to be restricted outside the mucus
layer, probably due to the charge repulsion between the negatively charged mucus and anionic LPS micelles
(Figure 6, upper panel). The mucolytic agent decreases the viscosity of mucus layer which makes it easy to
detach from the epithelial surfaces [32]. Therefore, the FITC-LPS administered with the mucolytic agent was able
to infiltrate the mucus layer and had access to the epithelium surfaces, which lead to its absorption in to the
systemic circulation, as indicated by its presence on the basolateral side of villi (Figure 6, lower panel). It has also
been reported that the intestinal mucus layer is rich in LPS binding proteins [33], which might contribute to the
inability of LPS to penetrate the mucus layer.

Figure 7 shows the CLSM images acquired from the rats administered with FITC-LPS followed by
administration of Cy3-insulin loaded NPs. As expected, the Cy3-insulin was able to traverse the mucus layer
and underlying epithelium, as indicated by its presence on the basolateral side of villi (magenta). In contrast,
the FITC-LPS was still restricted away from the epithelial cell surfaces (green) by the mucus layer (red). These
results supported our hypothesis that the paracellular permeation enhancement effect of CS-NPs was specific for
the loaded insulin.
3.7. In vivo toxicity study

LPS is a potent proinflammatory agent, which play an important role in the

pathogenesis of endoxic shock [11]. It can initiate the production of noxious
mediators like acute-phase proteins, cytokines and prostaglandins, which are
responsible for the endotoxic shock. However, under the experimental conditions the
LPS can induce pathological processes only when it is administered parenterally
[34]. Therefore, it was decided to study whether the test NPs can promote the toxic
effects of orally administered LPS. To study this, the changes in body weight and the
blood biochemistry parameters (alanine aminotransferase and aspartate
aminotransferase) were evaluated.

As expected, the daily IP injections of LPS (5 mg/mL) lead to a significant decrease in body weight and increase in
the SGPT levels. In contrast, the oral administration of LPS (5 mg/kg) did not produce any adverse effects as
compared to the untreated controls (Figures 8a and 8b). It is a well-known fact that the LPS is poorly absorbed
from the intestine. It was reported that the large doses of orally administered LPS failed to produce any toxic
symptoms in rats [35]. In another study, the effects of orally administered LPS on the intestinal morphology and
cell proliferation were examined in rats. It was found that, there was no evidence of epithelial changes, leukocyte
infiltration or any other abnormality in the gut of LPS-treated as compared to the controls [11]. Similarly, the group
receiving oral LPS (5 mg/kg) followed by test NPs (10 mg/kg) did not show any significant changes in the body
weight, SGOT or SGPT levels as compared to the control. These results confirmed that the paracellular
permeation enhancement by CS-NPs does not promote the intestinal absorption of LPS.

5. Conclusions
The purpose of this study was to determine whether the CS-NPs can enhance the oral absorption of LPS (a model
toxin) present in the intestinal lumen. The biodistribution study indicated that orally administered LPS was unable
to enter the systemic circulation, whereas, the insulin loaded in NPs could be absorbed. Additionally, the toxicity
study confirmed that CS-NPs did not improve the oral absorption of LPS. On the basis of these results, it can be
concluded that the CS-NPs can be used as a safe carrier for the oral protein delivery.

Acknowledgment
This work was supported by a grant from the National Science Council (NSC 96-2120-M-007-004), Taiwan,
Republic of China.

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