Biomaterials xxx (2013) 1e10

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Intestinal mucosa permeability following oral insulin delivery using

core shell corona nanolipoparticles
Xiuying Li a, b, Shiyan Guo a, Chunliu Zhu a, Quanlei Zhu a, Yong Gan a, *, Jukka Rantanen b,
Ulrik Lytt Rahbek c, Lars Hovgaard d, Mingshi Yang b, **
a
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China
b
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, Denmark
c
ADME Department, Novo Nordisk A/S, Maalov 2760, Denmark
d
Oral Formulation Development, Novo Nordisk A/S, Malov 2760, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: Chitosan nanoparticles (NC) have excellent capacity for protein entrapment, favorable epithelial
Received 28 July 2013 permeability, and are regarded as promising nanocarriers for oral protein delivery. Herein, we designed
Accepted 19 August 2013 and evaluated a class of core shell corona nanolipoparticles (CSC) to further improve the absorption
Available online xxx
through enhanced intestinal mucus penetration. CSC contains chitosan nanoparticles as a core compo-
nent and pluronic F127-lipid vesicles as a shell with hydrophilic chain and polyethylene oxide PEO as a
Keywords:
corona. These particles were developed by hydration of a dry pluronic F127-lipid film with NC sus-
Oral protein delivery
pensions followed by extrusion. Insulin nested inside CSC was well protected from enzymatic degra-
Core shell corona
Insulin
dation. Compared with NC, CSC exhibited significantly higher efficiency of mucosal penetration and,
Mucus penetrating particles consequently, higher cellular internalization of insulin in mucus secreting E12 cells. The cellular level of
Cellular uptake insulin after CSC treatment was 36-fold higher compared to treatment with free insulin, and 10-fold
higher compared to NC. CSC significantly facilitated the permeation of insulin across the ileum
epithelia, as demonstrated in an ex vivo study and an in vivo absorption study. CSC pharmacological
studies in diabetic rats showed that the hypoglycemic effects of orally administrated CSC were 2.5-fold
higher compared to NC. In conclusion, CSC is a promising oral protein delivery system to enhance the
stability, intestinal mucosal permeability, and oral absorption of insulin.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction from degradation in harsh pH environment, by enzymes in the

Despite rapid progress made in the development of modern
drug delivery technologies, an efficient oral delivery of therapeutic
proteins and peptides remains to be achieved. Oral delivery of
protein drugs faces several barriers, including pre-systemic
degradation, limited mucosal diffusion, and poor intestinal
epithelial membrane permeability [1e3]. A variety of innovative
approaches have been developed to tackle these challenges,
including the use of small molecule permeation enhancers, enzyme
inhibitors, and the encapsulation of protein drugs into micro-
spheres or nanoparticles [4,5]. The development of nanoparticles
with biodegradable and non-toxic polymers has become one of the
major focal areas in this field due to their ability to protect proteins
* Corresponding author. Tel.: þ86 21 20231000 1424; fax: þ86 21 20231000 1425.
** Corresponding author. Tel.: þ45 35336141.
E-mail addresses: simm2122@vip.sina.com (Y. Gan), mingshi.yang@sund.ku.dk
(M. Yang).
gastrointestinal (GI) tract [6e9], and their ability to modulate
physicochemical characteristics, drug release, and biological
behavior [10,11].
Among those polymeric nanoparticles, chitosan and chitosan
derivative based nanocarriers (NCs), appear to be particularly
promising [12]. They can significantly enhance the oral absorption
of peptides [13] and have excellent capacity for protein entrapment
and low toxicity [14]. They enhance the oral absorption of proteins
by several mechanisms, such as bioadhesion with the negative
charged cell membrane, transient widening of tight junctions and
increasing cell permeability by affecting paracellular and intracel-
lular pathways without changing junctional morphology [15].
However, they cannot effectively transport therapeutic proteins
across the mucus barrier due to the electrostatic interaction be-
tween the anionic mucus gel and cationic nanoparticles [16]. It also
has been reported that the binding of NCs to the surfaces of
epithelial cells and subsequent absorption-enhancing effects are
significantly reduced by mucus [16e18]. Although the adhesion of

0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biomaterials.2013.08.048

Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048
2 X. Li et al. / Biomaterials xxx (2013) 1e10
NCs to mucus can slow particle transit time through the GI tract,
and thus enhance drug absorption, the absorption enhancement is
limited by mucus turnover [19].
Recent studies have revealed that nanoparticles coated with
hydrophilic polymers exhibit decreased adhesion to mucus com-
ponents [19]. Our previous work also shows that Pluronic F127
modified lipid vesicles have superior mucus penetration, compared
to unmodified lipid vesicles [20]. To further improve the mucus
penetration of chitosan nanoparticles, we attempted to design a
core shell corona nanolipoparticles (CSC), using chitosan nano-
particles as a core component and F127-lipid vesicles as a shell with
polyethylene oxide PEO chains as the corona. By enveloping the
F127-lipid shell, the positively charged chitosan nanoparticles (NC)
would be shielded, ensuring free diffusion of NC in mucus. Addi-
tionally, protein nested in the CSC core would be better protected
from enzymatic degradation, and therefore the efficacy of drug
delivery could be further enhanced. To the best of our knowledge,
the CSC particles, which were designed to feature advantages of
both NC and Pluronic F127-lipid vesicles, are the newly designed
nano-size lipoparticles for oral protein delivery with significantly
enhanced mucus penetration.
In the present study, insulin was chosen as a model biomole-
cule for testing the CSC system. Insulin has currently attracted
great interest in developing protein delivery approach, due to its
wide clinical use, but limited routes of administration. Herein, CSC
was prepared by hydration of a polymer-lipid film with NC sus-
pension to form coreeshell structure and they were then char-
acterized in terms of particle size, zeta potential, and morphology.
Their ability to protect the encapsulated insulin from enzymatic
degradation, mucus penetration property, and cell uptake effi-
ciency and permeability were evaluated in HT29-MTX-E12 (E12)
cells. Pharmacological and pharmacokinetic studies were con-
ducted in streptozotocin (STZ) induced diabetic Sprague-Dawley
rats.
2. Materials and methods
2.1. Materials
Protasan UP CL113 was purchased from Nova Matrix (Drammen, Norway). Egg
phosphatidylcholine (EPC) was purchased from Q.P. Corp (Tokyo, Japan). Pluronic
F127 was donated by BASF (Ludwigshafen, Germany). RPMI1640 medium, Alexa
Fluor-555-labeled wheat germ agglutinin and 0.25% trypsin-0.53 mmol/L EDTA
were purchased from Invitrogen (Ontario, Canada). Paraformaldehyde was pur-
chased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2-(4-
amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from
Beyotime Institute of Biotechnology (Jiangsu, China). All the other reagents were of
analytical grade. Alexa 488 labeled insulin was synthesized by Novo Nordisk A/S
(Copenhagen, Denmark). HT29-MTX-E12 (E12) cells (52nde56th passages) cultured
for 14e18 days were supplied by the ADME Department of Novo Nordisk A/S. Caco-2
cell lines were obtained from the American Type culture collection (ATCC, Manassas,
VA, USA). Cells from the 40th 43rd passages were used in the present study.
2.2. Preparation and characterization of nanocarriers
2.2.1. Preparation and characterization of chitosan nanoparticles
Chitosan nanoparticles (NC) were prepared using a previously reported proce-
dure [21], based on the ionotropic gelation of chitosan with sodium tripolyphos-
phate (TPP) where the positively charged amino groups of chitosan interact with the
negative charged TPP. Briefly, TPP (1.0 mg/mL) was added to chitosan solution
(1.0 mg/mL) under magnetic stirring at room temperature to produce NC at a final
chitosan/TPP weight ratio of 6:1. The insulin-loaded NC were obtained by mixing the
insulin solution in 0.01 M NaOH (3.75 mg/mL) with NC solution at a theoretical
content of 30% (w/w) of insulin. Self-assembled NC were collected and washed
thrice with distilled water by centrifugation at 16,000 g on a 10-mL glycerol bed for
30 min. The centrifuged NC were then re-dispersed in distilled water and stored at
4  C until use.
The association efficiency and loading capacity of insulin in NC were determined
by subtracting the amounts of free insulin in supernatants quantified using high
performance liquid chromatography (HPLC) from the amounts added, using the
following equation:
Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048
total amount of insulin added  free insulin
Association efficiency ¼ (1)
total amount of insulin added
total amount of insulin added  free insulin
Loading capacity ¼ (2)
weight of nanoparticles
2.2.2. Preparation of core shell nanolipoparticles
The homogeneous F127-lipid film was prepared by drying a chloroform solution
containing egg phosphatidylcholine with F127 (4:1, mol/mol) and then hydrating
the film with NC suspensions (lipids/NP ¼ 6:1, w/w) for 30 min at room temperature,
followed by 6 rounds of extrusion through a polycarbonate membrane with 200-nm
pores (Avestin Inc., Canada). As a control, core shell nanolipoparticles (CS) without
hydrophilic corona was prepared by encapsulating NC in a pure lipid vesicle without
F127.
2.2.3. Characterization of nanocarriers
The mean particle sizes and zeta potential values of different nanocarriers were
measured using a Malvern Zetasizer NanoZS (Malvern Instruments, London, U.K.).
Each measurement was made in triplicate. A transmission electron microscope
(TEM, CM-200, Philips, Netherlands) was used to observe their morphology. The
samples were stained with an aqueous solution of phosphotungstic acid (1%, w/v)
before observation and the images were taken at 160 kV. The stability of these
nanocarriers in stimulated gastric fluid (SGF) and stimulated intestinal fluid (SIF)
were tested by measuring the changes in the particle size and polydispersity index
(PDI) after a 2-h incubation.
2.3. In vitro enzymatic degradation assay
The stability of nanocarriers against enzymatic degradation was carried out
using a procedure described previously [22]. In brief, trypsin (2500 IU/mg) and
chymotrypsin (800 IU/mg) were dissolved in Tris buffer (pH ¼ 8.0) containing 1 mM
CaCl2 at final concentrations of 250 IU/mL and 50 IU/mL, respectively. Each enzyme
solution (50 mL) was separately incubated with 950 mL of test formulations (NC and
CSC) containing 5 IU/mL of insulin. These formulations were pre-incubated at 37  C
for 15 min. 50 mL of each sample was taken at pre-determined intervals and the
enzyme activity was terminated by addition of 50 mL ice cold acetonitrile solution
containing 0.1% trifluoroacetic acid. Samples were subsequently treated with Triton
X-100 to remove the CSC and CS lipid shell to release insulin. The samples were then
analyzed using HPLC to determine the amount of insulin. Plain insulin solution and
insulin-loaded NC suspensions were used as control and treated samples under the
same experimental conditions.
2.4. Cell-based assays
2.4.1. Cytotoxicity of nanocarriers
Cytotoxicity of various nanocarrier suspensions in Caco-2 cells was evaluated
using the MTT assay. Briefly, Caco-2 cells were seeded onto 96-well plates at a
density of 1.0  104 cells per well. After a 48-h culture, nanocarrier suspensions were
added to the culture media. After a 2-h incubation at 37  C, the suspensions were
replaced by 100 mL of MTT solution (0.5 mg/mL in HBSS, pH ¼ 7.4) and incubated for
an additional 3 h at 37  C. Subsequently, the MTT medium was removed and 150 mL
of DMSO was added to dissolve the formazan crystals. The absorbance of the
resultant solutions was measured at 490 nm using a microplate reader (Bio-Rad,
USA). Each sample was analyzed in five replicates.
2.4.2. Transport study in E12 cell monolayers
The transport study was conducted following a previously reported procedure
[23]. The E12 cell monolayers were washed thrice with pre-warmed 1 Dulbecco’s
phosphate buffered saline (PBS, pH 7.4) and equilibrated in the 1 HBSS (with Ca2þ
and Mg2þ, 25 mM D-glucose) at 37  C and 95% relative humidity for 30 min. Alexa 488
insulin stock solution (0.5 mg/mL) and the Alexa 488 insulin loaded nanocarrier
suspensions were diluted with PBS. On the day of the experiment, the donor (apical)
solutions were prepared by diluting an aliquot of these solutions into HBSS to make
a final Alexa 488 insulin concentration of 40 mg/mL. For all the transport experi-
ments, a total of 200 mL of each acceptor (basolateral) sample was removed at 0, 15,
30, 45, 60, or 90 min after drug administration. For each acceptor sample taken,
200 mL of fresh HBSS was added to the mixture to maintain a constant volume. The
samples were transferred onto a 96-well titer plate and Alexa 488 insulin was
detected using a microplate fluorometer (Model 680, Bio-Rad, USA) at an excitation
filter of 485 nm and an emission filter of 530 nm. To eliminate the effects of mucus
on the transport of free Alexa 488 insulin, the E12 cells were incubated with 10 mM
N-acetyl cysteine (NAC) in HBSS for 30 min to remove the mucus prior to the
transport study. Apparent permeability coefficient (Papp) and enhancement ratio (R)
were calculated using the following equations.
dQ 1
Papp ¼  (3)
dt A  C0
 X. Li et al. / Biomaterials xxx (2013) 1e10
Table 1
Particle size, polydispersity index (PDI), z-potential, association efficiency (AE), and loading efficiency (LE) of NC, CS, and CSC.
Formulations Mean Diam (nm) PDI
NC 210.5  45.3 0.311  0.075
CS 202.8  22.9 0.175  0.069
CSC 195.3  32.9 0.151  0.048
Papptest
R ¼
Pappcontrol
Where dQ/dt is the flux of insulin from the apical side to the basolateral side, C0 is the
initial concentration of insulin in the apical compartment, and A is the membrane
area (cm2). Papp-test is the Papp of different groups, while Papp-control is the Papp of those
treated with insulin solutions.
2.4.3. Mucus penetration in E12 cells analyzed by confocal microscopy
In order to assess the interactions between Alexa 488 insulin and the mucus, the
E12 cells grown on TranswellÔ filter inserts for 14e17 days were treated as
described above. The apical incubation buffer, containing Alexa 488 insulin loaded
nanocarriers, was removed after 60 min of incubation with the formulations. The
E12 monolayers were then washed with PBS and the mucus layer was stained with
Alexa Fluor 555 labeled wheat germ agglutinin (Alexa Fluor 555-WGA, 10 mg mL1)
for 10 min at 37  C [24]. The membranes supporting the cell layers were washed
with PBS, and the cell layers supporting membranes of Transwell inserts were cut
from the plastic support without fixation, mounted onto microscope slides, and
covered with coverslips. The slides were immediately observed under a confocal
microscope (LSM 5 Pa, Zeiss, USA) using a 63 oil objective lens. Alexa Fluor 488 and
Alexa Fluor 555 were excited with a 488 nm argon laser and 543 nm helium-neon
laser, respectively. Image visualization and processing were performed using the
LSM 5 Pa software. To observe the interactions between the different formulations
with mucus in a larger view, a 2D image in the middle of the mucus was taken under
a confocal microscope at 5 magnification.
2.4.4. Qualification of mucus entrapment and cellular uptake of Alexa 488 insulin
To differentiate Alexa 488 insulin trapped in the mucus from bound insulin and
insulin that had been taken up by cells, the E12 cells were treated with the different
particle formulations for 1 h. The mucus layer was removed by washing the cell
monolayers twice with 300 mL of 4% (v/v) formalin solution in PBS (0.1 M, pH 7.4),
followed by the washing procedure as reported previously [17]. Then the cell layers
were disrupted with lysis buffer (RAPI), and cell-associated fluorescent protein was
determined by fluorescence spectroscopy. The amount of cell-associated Alexa 488
labeled insulin was reported as a percentage (%) of the initial amount of Alexa 488
labeled insulin.
2.5. Animal study
2.5.1. Animal care
Male Sprague-Dawley rats, 6e8 weeks old, were provided by the Animal
Experimental Center of Shanghai Institute of Materia Medica, Shanghai, China. The
animals had free access to rat chow and tap water. All of the animal experiments
were carried out according to the Institutional Animal Care and Use Committee
(IACUC) guidelines of Shanghai Institute of Materia Medica.
2.5.2. Absorption studies in the ligated intestinal loops
2.5.2.1. Permeation studies ex vivo. The transport of Alexa 488 insulin loaded CSC and
NC across the epithelial mucosa of rat ileum was monitored ex vivo, as described by
Yin et al. [25]. Briefly, after rats were sacrificed, 5-cm sections of the ileum were
excised and incubated in 10 mL Kreb’-Ringer buffer at 37  C with gentle agitation. CSC
and NC (0.5 mL) were loaded into the loops using a syringe. At various times (0.5, 1, 1.5
and 2 h), 200 mL of the incubation buffer was collected for determination of Alexa 488
labeled insulin using fluorescence spectroscopy. The same volume of fresh buffer was
added at each time point. All of the experiments were performed in triplicates.
2.5.2.2. Absorption assay in vivo. The in vivo uptake of CSC and NC were evaluated
using the ligated intestinal loops model following the method described by Yun et al.
[9]. Sprague-Dwaley rats were fasted overnight before experiments, but allowed free
access to water. The rats were anesthetized with pentobarbital sodium (0.04 mg/kg).
After the abdomen was exposed, 5-cm loops of ileum were made by ligation at both
ends and the tissue was washed with physiological saline. CSC and NC (0.5 mL,
100 mg/mL of Alexa 488 insulin) were injected into the loop. To study the effect of
mucus on the absorption of free Alexa 488 insulin, ligated ileum loops were pre-
incubated with saline containing 10% N-acetyl cysteine (NAC) for 10 min before
the absorption study to remove mucus, and then 0.5 mL CSC and NC suspensions
were injected into the loop. Rats were sacrificed after 0.5 h or 2 h and the sections of
each loop were removed and extensively washed using PBS. Subsequently, the
removed loops were fixed by 4% paraformaldehyde for 2 h and immersed in 30%
sucrose at 4  C overnight. Samples were embedded and frozen at 20  C (OCT,
Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048
3
z potential (mV) AE LE
þ36.6  4.5 76.6  5.8% 39.0  4.2%
7.1  3.2
4.3  5.4
Sakura Fine technical Co., Ltd.). Frozen ileum sections (20 mm) were cut using a
(4)
cryostat (Leica CM 1950) and then stained with DAPI. The tissue-sections were then
visualized using a confocal microscope.
2.5.3. Pharmacological and pharmacokinetic studies
Diabetes was induced in Male Sprague-Dawley rats weighting 180e200 g by an
injection of streptozotocin (65 mg/kg) dissolved in citrate buffer as previously
described [26]. The blood glucose level was determined using a glucose meter (On
CallÒ EZ II, Acon Biotech.Co.Ltd., Hangzhou, China). Rats were considered to be
diabetic when their fasting blood glucose level was higher than 250 mg/dL one week
after streptozotocin treatment. Sprague-Dwaley rats were fasted overnight before
experiments, but allowed free access to water. Various formulations of insulin were
administered to the diabetic rats by gavage of insulin solution (50 IU/kg), insulin
loaded NC (50 IU/kg), or insulin loaded CSC (50 IU/kg) or by subcutaneous injection
(SC) of insulin solution (5 IU/kg). Blood samples were collected from the tail veins of
rats prior to drug administration and at various times (1, 2, 4, 6, 8 and 10 h) after
dosing. The blood glucose levels were analyzed and the area under concentration-
time curve (AUC) over 12 h was calculated for each group. For the analysis of
serum insulin level, blood samples were centrifuged (3000 rpm, 5 min) and sub-
sequently quantified using an appropriate insulin ELISA kit (R&D system, Inc, MN,
USA). The total decrease (D%) in serum glucose levels were calculated using a
modified method as follows:
AUCðInsulin solutionÞ  AUCðtestÞ
D% ¼  100 (5)
AUCðInsulin solutionÞ
The relative bioavailability (F%) of CSC after oral administration was calculated
using the following formula.
AUCðoralÞ  DoseðscÞ
F% ¼  100 (6)
AUCðscÞ  DoseðoralÞ
2.6. Statistical analysis
Comparisons between the two groups were performed using Student’s t-test
(SPSS, Chicago, IL) and P < 0.05 was considered statistically significant.
3. Results
3.1. Characterization of nanocarriers
As shown in Table 1, the naked NC were around 210  45 nm in size
and had a positive zeta potential of approximately þ36.6 
4.5 mV. The mean association efficiency of insulin to NC was
76.6  5.8%, and the mean loading efficiency was 39.0  4.2%. The
incorporation of NC into vesicles did not have a remarkable effect on
the size, but led to a significant changes in zeta potential (P < 0.05),
which were reduced to e 7.1  3.2 and e 4.3  5.4 mV for CS and CSC,
respectively. The conversion of the zeta potential suggested that the
NC were encapsulated in the vesicles. As shown in Fig. 1, the core shell
corona structures were observed for CSC under TEM, indicating that
chitosan nanoparticles were encapsulated into the vesicles under the
current experimental conditions. A schematic presentation of the
core shell corona structure is shown in Fig. 1A. CSC formed by this
method remained stable in SGF and SIF, and the size and PDI did not
change significantly. CS was stable in SGF but the size greatly
increased after a 2-h incubation in SIF. Whereas, NC was soluble in SGF
and no particle size change could be observed in SIF (Data not shown).
3.2. Protection of insulin against enzymatic degradation
No significant difference was observed between CS and CSC with
respect to protection of insulin from enzymatic degradation by
trypsin and a-chymotrypsin. As shown in Fig. 2, CS and CSC
4 X. Li et al. / Biomaterials xxx (2013) 1e10

Fig. 1. Design and evaluation of NC and CSC. A: Schematic illustration of the formation of NC and CSC; B: TEM images of NC and CSC. Red line, the boundary between NC and
polymer-lipid shell. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

protected 40% and 70% of encapsulated insulin, respectively, from
degradation by trypsin and a-chymotrypsin after 1 h of incubation
with the intestinal enzyme solutions in vitro, whereas plain insulin
was almost completely degraded under the same conditions. This
suggested that the core shell structures could prevent insulin from
being exposed to the enzymes by shielding the protein in the core
of the nanolipoparticles. NC alone, in this case, failed to protect the
associated insulin from enzymatic degradation. Its degradation
profiles were similar to those of naked insulin.
3.3. Results from studies with cell-based models
promote transport of insulin across the E12 cell monolayers. As
shown in Table 2, the Papp obtained with NC was quite similar to
those obtained with insulin HBSS solution. However, after NAC
treatment, the insulin Papp was increased 2-fold, indicating that the
mucus formed a barrier for the transport of insulin. The use of CSC
was found to significantly enhance insulin Papp across the E12
monolayers. An R-value of 2.42 was obtained with CSC. This was
comparable to the results obtained using plain Alexa 488 insulin
HBSS solution in E12 monolayers after mucus depletion by NAC.
The unmodified CS also enhanced insulin transport to a less extent
as compared to CSC, with an R-value of 1.63.

3.3.1. In vitro cytotoxicity
As shown in Fig. 3, all the three nanocarriers were found to have
no significant cytotoxicity (P > 0.05) at the tested concentrations
(0.2, 0.5 and 1.0 mg/mL), although there was a slight reduction in
cell viability treated with 1.0 mg/mL of NC.
3.3.2. Transport of Alexa 488 insulin loaded nanocarriers across E12
cells
The apparent permeability coefficient (Papp) and enhancement
ratio (R) were used to assess the ability of the nanocarriers to
3.3.3. Mucus penetration of Alexa 488 insulin in E12 cells
In order to determine the mucus penetration properties of Alexa
488 insulin, the E12 cell monolayers treated with different for-
mulations were imaged without fixation to show the diffusion of
Alexa 488 insulin. The red fluorescence derived from Alexa Fluor
555 represents the mucus that covers the surface of the cell, and the
green fluorescent spots derived from Alexa 488 correspond to Alexa
488 insulin. Few green fluorescent spots were observed in the
monolayer treated with Alexa 488 insulin HBSS solution, but
stronger green fluorescence was observed in monolayers treated

Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048
 X. Li et al. / Biomaterials xxx (2013) 1e10 5

A 100 B 100 S CS
S CS NC CSC
NC CSC
80 80

% undegradated insulin
% undegradated insulin

60 60

40 40

20 20

0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120
Time (min) Time (min)

Fig. 2. Enzymatic degradation profiles of insulin as a function of time A: Trypsin; B: a-chymotrpsin.

with NC, CS, and CSC (Fig. 4A). After NC treatment, the mucus layer
was discontinuously distributed and showed strong yellow spots,
but the mucus layers treated with CS and CSC were similar to those
treated with Alexa 488 insulin solution, except for stronger green
fluorescence. The 2D images from the middle position of the mucus
on the surface of the E12 cell monolayer (Fig. 4B) showed that NC
closely interacts with mucus, forming aggregates with some of its
components (big yellow spots) and some NC self-aggregates (large
green spots). This phenomenon was seen much less in E12 cell
monolayers treated with Alexa 488 insulin loaded CS and CSC and
the aggregates were much smaller than those seen in the NC group.
3.3.4. Concentrations of Alexa 488 insulin in the mucus layer and
cell layers
To differentiate the fraction of Alexa 488 insulin trapped in the
mucus layer and the fraction associated with, or internalized in the
cells, the mucus layer was washed away using the formalin solu-
tion. As shown in Fig. 5, the CSC had the strongest interaction with
E12 cells, reaching a maximum of about 9.3% of the initial Alexa 488
insulin in the mucus layer and cells. Among these particles, 5.4%
were inside in or attached to the E12 cells. The CS had an effect
similar to that of the CSC, with 8.6% of the initial Alexa 488 insulin
in the mucus layer or the E12 cells, but only 1.9% was inside or
attached to the E12 cells, far less than that with the CSC. The NC also
120
NC
100
80
Cell Viability (%)
showed stronger association with the entire cell monolayer
compared to the insulin solution, as suggested by the observation
that 4.3% of the initial Alexa 488 insulin was detected in the E12
monolayers prior to removal of mucus, but only 0.5% of the initial
Alexa 488 insulin was also detected inside the cell. These results
suggested that the enhanced association efficiency of insulin with
cells was not as significant as the association with the entire cell
monolayer with intact mucus.
3.4. Results from the animal studies
3.4.1. The accumulative transport of insulin ex vivo
The transport of NC and CSC across the intestinal epithelia was
investigated using ex vivo in ligated ileum loops. As shown in Fig. 6,
compared to NC, CSC increased the accumulative amount of Alexa
488 insulin permeated through the rat ileum. The permeated Alexa
488 insulin from CSC at 1 h was 21.75  0.86 ng, which was 1.53-
fold higher than that from NC (14.18  0.59 ng) and the accumu-
lative amount of Alexa 488 insulin at 2 h increased to
61.74  7.39 ng, which was 1.76-fold higher compared to NC. These
results revealed that absorption enhancement of CSC across the
epithelia was associated with improved mucus penetration and
cellular uptake.
3.4.2. Absorption studies in villi
The absorption of NC and CSC was qualitatively observed by a
CS CSC confocal microscope to be located in the villi of ileum loops. Fig. 7
shows the absorption of Alexa 488 insulin from NC and CSC at 0.5
and 2.0 h, respectively. The absorption of Alexa 488 insulin
increased with time, and stronger green fluorescence was observed
in loops treated with CSC. Alexa 488 insulin loaded CSC permeated

deeply into villi after 2 h, indicating their effective absorption

in vivo. With NAC pre-treatment to remove the mucus, both NC and
60 CSC showed strong green fluorescence intensity. The effect of

Table 2
40 Apparent permeability coefficient (Papp) and enhancement ratio (R) of insulin
permeability across E12 cell monolayers. * P < 0.05, compared with S group.

20 Formulations Papp*107(cm/s) Enhancement ratio

S 4.73  0.35 e
S þ NAC 7.71  0.72* 2.21
0
0.5 mg/mL 1.0 mg/mL NC 4.58  0.53 0.97
Control 0.2 mg/mL
CS NP 7.29  0.89* 1.63
CSC NP 11.46  1.58* 2.42
Fig. 3. Effect of different formulations on the Caco-2 cell viability.

Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048
6 X. Li et al. / Biomaterials xxx (2013) 1e10

Fig. 4. Mucus and cell-based evaluation of NC and CSC. A: Confocal microscope images of E12 cell layers treated with different formulations. A: 3D image of mucus penetration. B:
2D images of E12 monolayers. Red: mucus covering the cell surface stained with Alexa Fluor 555-WGA. Green: Alexa 488-insulin. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048

Insulin bound to and within cells and mucus
12
mucus+cell * ##
cell
10
8
(% of initial)
6
4 shell corona structured nanolipoparticles (CSC), based on a ‘parti-
2
0 in the polymer nanoparticle cores, enhancing mucus-penetrating
S NC
Fig. 5. Relative amounts of Alexa 488 insulin internalized/attached in E12 cells.
*P < 0.05, compared with NC group; #P < 0.05, compared with NC group; ##P < 0.01,
compared with the NC group.
mucus on the absorption of insulin loaded into NC was much more
significant than those loaded into CSC. For the NC group, the green
fluorescence was much stronger with NAC treatment, whereas the
difference after NAC treatment was less for the CSC group.
3.4.3. Hypoglycemic effect in vivo
The pharmacological effects of NC and CSC were evaluated in
diabetic rats after oral administration. As shown in Fig. 8A, the plain
insulin solution failed to reduce the blood glucose level; NC slightly
reduced the blood glucose level; and CSC exhibited stronger hy-
poglycemic effects than the other two treatments. Compared with
the baseline, the blood glucose level decreased to 77% and the
hypoglycemic effect lasted 10 h. The D% of CSC was 2.52-fold higher
compared to NC. The serum insulin concentration-time profiles are
shown in Fig. 8B. The rats subcutaneously treated with plain insulin
solution resulted in a rapid increase in serum insulin concentration,
whereas oral administration of CSC demonstrated a slower rise in
serum insulin concentration, reaching a maximum serum concen-
tration at 4 h after treatment. Compared to NC and plain insulin
solutions, CSC treatment showed significant higher serum insulin
75
NC
Amount of insulin permeated (ng)
CSC
50
25
0 mucus. In addition, the Papp value of insulin solution was signifi-
0.5 1.0
Time (h)
Fig. 6. Accumulative permeated amount of Alexa 488 insulin across rat ileum from NC
and CSC (Mean  SD, n ¼ 3).
Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048
X. Li et al. / Biomaterials xxx (2013) 1e10 7
concentrations at 1e6 h post administration. As shown in Table 3,
* the AUC(0e12 h) for CSC was 232.5  35.5 mIU*h/mL, with a relative
bioavailability of 7.8%; both parameters were significantly greater
# compared with NC and plain insulin.
4. Discussion
Pre-systemic enzymatic degradation and poor transmucosal
permeability are the major obstacles to an efficacious oral protein
and peptide delivery [3], which can be overcome by the use of
nanoparticle carriers [7,10]. In the current study, we designed core
cle-in-particle’ approach. The system was comprised of chitosan
nanoparticles as a core component and F127-lipid vesicles as a
shell, with hydrophilic chain polyethylene oxide (PEO) as a corona.
These particles protect fragile biomacromolecules, such as insulin,
CS CSC properties and membrane transportation. CSCs were prepared by
hydration of polymer-lipid dry film with NC suspensions. NC were
designed to be covered by the vesicles via hydrogen bonds between
the particle surface and lipid polar heads and electrostatic in-
teractions between lipids and the hydrophilic surface [27]. As ex-
pected, most NC particles were entrapped in the vesicle as observed
by TEM, where the dense black cores of NC were surrounded by
gray lipid shells (Fig. 1B). The sizes of CSC were around 200 nm, as
shown in TEM. This was also in close agreement with the particle
sizes indicated by dynamic light scattering measurements. Upon
adsorption of lipid or polymer-lipid layers onto NC, the strong
positive zeta potential of NC changed from þ36 mV to negative
charges (7.1 and 4.3 mV, respectively), which also showed that
the cores of NC were encapsulated into the polymer-lipid vesicles
and formed nanolipoparticles. Similar assembly of nanoparticles
and vesicles has been reported by other groups [28,29]. However,
most other carriers reported in the literature are macro-sized and
do not feature a hydrophilic polymer chain on the surface, which
are unstable in the GI tract and are not ideal for intestinal
absorption.
Fragile biomacromolecules, such as insulin, packaged in the
nanolipoparticles with solid cores are expected to be able to
withstand the harsh physiologic environment due to the shielding
shell of the polymer-lipid layers, preventing direct contact of the
biomacromolecules with enzymes. Our in vitro forced degradation
study confirmed this hypothesis. The naked NC failed to protect
insulin from enzymatic degradation; whereas, the enzymatic
degradation of insulin associated with chitosan nanoparticles was
significantly decreased when they were encapsulated in vesicles.
These results suggested that nanolipoparticles with core shell
structures may improve the integrity of fragile biomolecules during
oral delivery.
Whether mucus is also a barrier to the diffusion of protein and
peptide drugs is still debatable. The traditional theory is that the
pore size of mucus physically constrains the diffusion of macro-
molecules [30,31]. However, in recent years, some emerging evi-
dence has supported the opposite theory, that mucus is not a
diffusion barrier for protein drugs [32,33]. In our study, only very
little Alexa 488 insulin was detected in the mucus. As shown in the
confocal microscope images (Fig. 4A), very weak green fluorescence
was visible, indicating that it was difficult for Alexa 488 insulin to
pass through the mucin network. The qualitative data (Fig. 5) also
confirmed that minimal Alexa 488 insulin was present in the
1.5 2.0 cantly increased after the mucus was removed by NAC treatment.
These results supported the notion that mucus is a diffusion barrier
to insulin and that it is necessary to consider drug delivery strate-
gies that would overcome the physical barrier of the mucus layer.
8 X. Li et al. / Biomaterials xxx (2013) 1e10
Fig. 7. Distribution of Alexa 488 insulin loaded in NC or CSC in ileum villi at 0.5 and 2 h. Green fluorescence refers to Alexa 488 insulin and blue fluorescence refers to cell nucleus.
It has been reported that the absorption of insulin can be
enhanced by using fusogenic liposomes (FLs) as carriers when they
are directly administered to colonic and rectal loops [34]. However,
the enhancement in absorption is significantly decreased when
administered to the ileum [34]. It has been speculated that the
mucus layer overlying the ileal epithelia may impair or compromise
the fusion of FL to the intestinal mucosa. It also has been reported
that trimethyl chitosan (TMC) and chitosan do not enhance the
absorption of insulin in the ileum because of the thick mucus layer
[35]. In the current study, we investigated the mucus-penetrating
properties of chitosan nanoparticles, which have strong electro-
static interactions with mucus. Strong green fluorescence was
observed in the mucus network of E12 cells after NC treatment,
suggesting that NC could increase the amount of insulin trapped in
mucus. However the ability of NC to enhance cellular uptake and
cellular transport was less significant than its ability to enhance
mucus entrapment. These results suggested that NC could not
readily improve the absorption. This could be because most of the
NC is trapped in the mucus due to the strong electrostatic in-
teractions between chitosan and mucin [16]. NC were unable to
reach the epithelial surface, and so failed to improve the absorption
of encapsulated proteins. Considering the mucus turnover in vivo,
such a protein delivery system could be rapidly cleared together
with the mucus. Therefore, an ideal pharmacological efficacy will
be difficult to obtain. Unlike NC, CSC not only significantly
enhanced the mucus penetration of insulin but also greatly
enhanced the efficiency of cellular associations. As shown in the
LSM images, the mucus of E12 cells treated with CSC was homo-
genously distributed. The cell monolayers treated with CSC showed
strong green spots, indicating that more protein molecules were
able to reach the cell surface and promote cellular uptake.
In addition to their ability to enhance mucus penetration, CSC
may also enhance cellular uptake of insulin by surface modification
of the nanolipoparticles with F127 polymers. F127 has been re-
ported to stabilize and seal damaged lipid bilayer membranes [36e
38]. This may reinforce the association of the nanolipoparticles
with the cell membrane. The increased level of cellular insulin
Please cite this article in press as: Li X, et al., Intestinal mucosa permeability following oral insulin delivery using core shell corona
nanolipoparticles, Biomaterials (2013), http://dx.doi.org/10.1016/j.biomaterials.2013.08.048
uptake observed with CSC in E12 cells showed 10-fold higher up-
take compared to NC. Although CSC have been found to improve
insulin transport through E12 cells as compared to insulin solution
and naked NC (Table 2), their impacts on the Papp values are not
comparable to their effects on the efficiency of cellular uptake.
Similar results have been reported by Anchalee et al. and Thompson
et al.: the chitosan nanocomplexes could improve cellular uptake of
insulin with E12 cells, but no insulin was detected on the baso-
lateral sides of the E12 cell monolayer [23,39].
In the present ex vivo transport study across ligated ileum loops,
we found that CSC significantly enhanced the permeability of Alexa
488 insulin compared with NC. When reaching the small intestine,
NC were mostly immobilized in the mucin network, but CSC could
penetrate through the mucus and thus more Alexa 488 insulin
could reach the epithelium surface and be transported across the
intestinal epithelium via the paracellular pathway, transcytosis or
receptor-mediated transcytosis. The absorption enhancement ef-
fect of CSC was further confirmed by in vivo absorption. Alexa 488
insulin could clearly be observed in the villi after 0.5 h treatment of
ligated ileum loops treated with CSC, while minimal Alexa 488
insulin could be seen in the NC group. The green fluorescence in the
CSC group was significant stronger compared to the NC group at 2 h
post administration of CSC. With NAC pre-treatment to remove the
mucus, ligated loops treated with NC showed significantly stronger
green fluorescence intensity compared to the non-NAC treated NC
group, while only slightly stronger fluorescence was observed in
the CSC group compared to non-NAC treated CSC group. These re-
sults indicated that the effect of mucus on the absorption of insulin
loaded in NC was much more significant than those loaded in CSC,
suggesting enhanced mucus penetration of NC by polymer-lipid
vesicle encapsulation.
The in vivo pharmacological and pharmacokinetic results
showed good correlations with the improved absorption and
transport of insulin in the ligated intestinal loop and cell studies
in vitro. As expected, the oral administration of plain insulin solution
did not lead to significant hypoglycemic effects, because of the rapid
degradation in the gastric fluid and poor permeability through
 X. Li et al. / Biomaterials xxx (2013) 1e10 9

A 120 S NC
administration and lasted for 10 h. During this experiment, the
fasting food blood glucose did not return to the initial level after
S-SC CSC 10 h, similar as other previous reports, which might be the conse-
Blood glucose level (% of initial)

100 quence of the combined effects of hunger and hypoglycemic agents

[9,21]. The quick absorption of insulin suggested the fast diffusion of
80 CSC through the mucosal barrier while the long-acting effect
revealed the slow release of insulin from the core shell corona
structure. Furthermore, the animals treated with CSC exhibited
60 significantly higher serum insulin levels with a significantly
increased relative bioavailability compared with the NC and free
40 insulin groups. In the clinical practice, sc. administration of insulin
can be associated with severe hypoglycemic shock and patient
20 inconvenience. Our results demonstrated that insulin encapsulated
in CSC exhibited prolonged hypoglycemic activity without the initial
hypoglycemia seen with sc. administration, suggesting that CSC may
0
be a promising strategy for clinical treatment of diabetes.
0 2 4 6 8 10 12
Time (h)
B 160 5. Conclusions
S NC
140 S-SC CSC In the present study, we designed core shell corona nano-
Serum insulin level (µIU/mL)

lipoparticles to improve the intestinal mucosal permeability of in-

120
100
80
60
40
20
0
0 2 4 6 8 10
Time (hour)
Fig. 8. A Blood glucose levels in diabetic rats following oral administration of insulin
loaded NC, CSC, and insulin solution (S, 50 IU/kg) and subcutaneous injection of insulin
solution (S-SC, 5.0 IU/kg) (Mean  SD, n ¼ 4). *P < 0.05, compared with S group;
**P < 0.01, compared with S group; #P < 0.05, compared with the NC group. B: Serum
insulin level in diabetic rats following oral administration of insulin loaded NC, CSC
and insulin insulin solution (S, 50 IU/kg) and subcutaneous injection of insulin solution
(S-SC, 5.0 IU/kg) as positive control (Mean  SD, n ¼ 4). *P < 0.05, compared with the
NC group.
epithelia. Insulin loaded NC had limited efficiency to reduce the
blood glucose level, partially because of the instability of NC as seen
in in vitro studies. Strong adhesion to the mucus, instead of pene-
trating through the mucus, also contributed to the ineffectiveness of
insulin loaded NC. Whereas, CSC exhibited improved stability in the
GI tract, enhanced mucus penetration, and membrane transport,
leading to significantly more potent and prolonged pharmacological
efficacies. The onset of hyperglycemic effect occurred at 2 h post
sulin. The insulin loaded chitosan nanoparticles formed the core,
and polymer-lipid vesicles formed the shell, with hydrophilic chain
PEO as the corona. Experiments performed with mucus-secreting
HT29-MTX-E12 cells suggested that mucus might constitute a
diffusion barrier for insulin molecules and prevent them from
reaching the cell membrane. The naked NC were found to increase
the amount of protein entrapped in the mucus, while CSC improved
the ability of insulin to penetrate the mucus and increased cellular
uptake of insulin. Enhanced permeability of insulin was confirmed
by a transport study using ex vivo intestinal tissue. Enhanced ab-
sorption of insulin in intestinal villi by CSC was also observed in
12 in vivo absorption study. The CSC treatment resulted in an elevated
serum insulin concentration and improved hypoglycemic effect in
diabetic rats, suggesting that NC encapsulated in F127-lipid vesicles
could be a promising nanocarrier for oral delivery of insulin, as well
as other peptides and proteins.
Acknowledgments
This work was supported by the Novo Nordisk-Chinese Academy
of Science (CAS) Research Foundation (No. NNCAS-2009-10) and
the National Science and Technology Major Project, “Key New Drug
Creation and Manufacturing Program” (2012ZX09301001-001). We
thank Erik Wisaeus and Pia Wahlberg of the Danish Technological
Institute, Denmark, for their excellent assistance on TEM, which is
supported by The Innovation Consortium NanoMorph (952320/
2009) funded by The Danish Council for Technology and Innova-
tion. We also thank the ADME department of Novo Nordisk A/S,
Denmark, for excellent assistance in HT29-MTX-E12 cell studies.
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