Research Article

Cite This: ACS Appl. Mater. Interfaces 2018, 10, 9315−9324 www.acsami.org

Novel Solid Lipid Nanoparticle with Endosomal Escape Function for

Oral Delivery of Insulin
Yining Xu,† Yaxian Zheng,† Lei Wu, Xi Zhu, Zhirong Zhang, and Yuan Huang*
Key Laboratory of Drug Targeting and Drug Delivery System (Ministry of Education), West China School of Pharmacy, Sichuan
University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041 Sichuan, China
*
S Supporting Information

ABSTRACT: Although nanoparticles (NPs) have been demonstrated

as promising tools for improving oral absorption of biotherapeutics,
most of them still have very limited oral bioavailability. Lyso−endosomal
degradation in epithelial cells is one of the important but often-neglected
physiological barriers, limiting the transport of cargoes across the
intestinal epithelium. We herein reported a solid lipid nanoparticle
(SLN) platform with a unique feature of endosomal escape for oral
protein drug delivery. The SLNs consisted of a solid-lipid shell, which
contained an endosomal escape agent (GLFEAIEGFIENGWEGMIDG-
WYG, HA2), and an aqueous core that is loaded with insulin (INS HA2-
O-SLNs). SLNs without and with the HA2 peptide in the aqueous core
(INS SLNs and INS HA2-W-SLNs, respectively) were used as the
control groups. Our study showed that INS HA2-O-SLNs effectively
facilitated the escape of the loaded insulin from the acidic endosomes,
which preserved the biological activity of insulin to a greater extent during the intracellular transport. The spatial location of the
HA2 peptide was demonstrated to determine the endosomal escape efficiency. As demonstrated in the intracellular trafficking of
SLNs, INS HA2-O-SLNs displayed much less distribution in late endosomes and lysosomes. Meanwhile, insulin in INS HA2-O-
SLNs exhibited the highest transepithelial permeation efficiency, with 2.19 and 1.72 folds higher accumulated amount in the
basolateral side as compared to that in INS SLNs and INS HA2-W-SLNs. In addition, insulin from INS HA2-O-SLNs exhibited
the highest insulin permeation in various regions of small intestines. INS HA2-O-SLNs generated an excellent hypoglycemic
response following oral administration in diabetic rats. Thus, such functional SLNs demonstrated a great potency for oral delivery
of peptide/protein drugs.
KEYWORDS: solid lipid nanoparticles, insulin, endosomal escape, HA2 peptides, oral delivery

1. INTRODUCTION the terminal enzymatic degradative compartments.12 This

Therapeutic peptides and proteins have been extensively
utilized in the treatment of numerous diseases for their
specificity and high potency.1 However, these biotherapeutics
are currently only limited to parenteral administration, which
result in unwanted pain, poor patient compliance, and several
side effects. Although oral administration is much more
preferred, bioavailability of these drugs is often limited by
several physiological barriers in the gastrointestinal tract
(GIT).2,3 Since 1980s, nanotechnology has been reported to
protect drugs from harsh conditions in the GIT.4−6 However, it
is still a big challenge to improve the drug permeability across
the intestinal epithelium.7−9
Endocytic pathway is the major route for most nanoparticle
(NP) uptake.10,11 However, the amount of NPs across the
intestinal epithelium was always much less than those engulfed
in cells. The dilemma of “easy endocytosis and hard
transepithelial transport” is one of the major problems that
hinders the therapeutic effect of proteins in circulation. After
internalization, these NPs are commonly entrapped in
endosomes and eventually degraded in lysosomes, which are
lysosomal degradation would drastically disrupt the protein
integrity and thus impeded the transport of drugs into blood
circulation.2 Therefore, lyso−endosomal entrapment is a
formidable but often-neglected barrier. An innovative and
practical strategy utilizing endosomal escape might be desired
for oral protein delivery.
To date, extensive efforts for endosomal escape have been
made on cancer therapies and gene transfer during the past
years.13−15 However, few research studies have been focused on
the nanoplatforms combining the endosomal escaping strategy
to improve the oral bioavailability of biotherapeutics.16 The
hemagglutinin-2 (GLFEAIEGFIENGWEGMIDGWYG, HA2)
peptide, derived from influenza virus coat, was introduced as a
nontoxic fusogenic agent. The protonation of the HA2 peptide
could induce a conformational change to α-helixes in response
to endosomal acidification and destabilize the membrane. The
Received: January 10, 2018
Accepted: February 27, 2018
Published: February 27, 2018

© 2018 American Chemical Society 9315 DOI: 10.1021/acsami.8b00507

ACS Appl. Mater. Interfaces 2018, 10, 9315−9324
ACS Applied Materials & Interfaces
contents were supposed to avoid lysosomal degradation, and
then, the transepithelial transport of drugs would be further
improved.17 Therefore, we hypothesized that a well-designed
nanocarrier with the HA2 peptide might held out a
considerable promise for oral delivery of biomacromolecules.
Herein, a novel delivery system was constructed with the
function of endosomal escape for orally administered protein
drugs, aiming to protect these drugs from lysosomal
degradation and enhance their oral bioavailability. The water
in oil in water (W/O/W) double emulsion was used to co-
encapsulate hydrophilic insulin (INS, model protein drug) and
the hydrophobic HA2 peptide (endosomal escape agent) in
solid lipid nanoparticles (SLNs). INS HA2-W-SLNs (HA2
peptide loaded in the inner aqueous phase of SLNs) and INS
HA2-O-SLNs (HA2 peptide mainly loaded in the outer oil
phase of SLNs) were respectively prepared and supposed to
have different release kinetics of the fusogenic agent. The effect
of spatial location of the HA2 peptide in SLNs was thoroughly
investigated via cellular uptake and intracellular trafficking.
Furthermore, transepithelial transport efficiency of formulations
was also conducted in vitro as well as ex vivo. Finally, in vivo
studies were performed in diabetic rats to elucidate the
pharmacological and pharmacokinetics of INS SLNs with the
HA2 peptide after oral administration.
2. MATERIALS AND METHODS
2.1. Materials. Porcine insulin was gained from Wanbang
Biochemical Co., Ltd. GLFEAIEGFIENGWEGMIDGWYG (HA2)
peptide was gained from Chinese Peptide Co., Ltd. Soybean lecithin
was supplied by Taiwei Pharmaceutical Co., Ltd. Glycerol tripalmitate
was gained from Alfa Aesar Co., Ltd. Pluronic F68 was gained from
Nanjing Weier Chemical Co., Ltd. Stearic acid was gained from
Huzhou Zhanwang Pharmaceutical Co., Ltd. Fluorescein isothiocya-
nate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC)
were gained from Sigma-Aldrich. Rabbit anti-Rab5 and mouse anti-
Rab7 antibodies were obtained from Abcam. LysoTracker Red was
purchased from Invitrogen. Other chemicals were of analytic grade.
2.2. Preparation and Characterization of SLNs. Insulin-loaded
SLNs (INS SLNs) as well as the two kinds of HA2 peptide and insulin
dual-loaded SLNs were produced by the micelle-double emulsion
method.18,19 As for the preparation of INS SLNs, 5 mg of insulin and
25 mg of sodium cholate were dissolved in pH 2 hydrochloric acid
(HCl) solution. Then, 1 mL of dichloromethane containing 15 mg of
soybean lecithin, 15 mg of tripalmitin, and 1 mg of stearic acid was
added. The mixture was emulsified by probe sonication for each
ultrasound 5 s, interval 5 s at 80 W within 2 min. Pluronic F68 solution
(4 mL; 2%, w/v) was then added and sonicated in the same way. After
eliminating the organic solvent by evaporation for 30 min at 37 °C,
this emulsion was diluted with Pluronic F68 solution. INS HA2-W-
SLNs or INS HA2-O-SLNs were prepared by using the aforemen-
tioned method, besides that the HA2 peptide was added in the inner
aqueous phase (INS HA2-W-SLNs) or oil phase of mixed solvents
(dichloromethane/methanol 9:1) (INS HA2-O-SLNs). Fluorescent-
labeled SLNs were prepared using FITC-labeled insulin (FITC-INS)
in the method as mentioned above.7,20,21 Meanwhile, TRITC and
FITC were used to fluorescently label INS and HA2 peptide,
respectively. The structures of the different SLNs were visualized
under confocal laser scanning microscopy (CLSM, Live 5 DUO, Carl
Zeiss, Jena, Germany). Various SLNs were characterized by Malvern
Zetasizer Nano ZS90. The size distribution was performed by
NanoSight LM10. Transmission electron microscopy (TEM, Hitachi
H-600, Tokyo, Japan) was applied to observe the morphologies of
SLNs.22 To evaluate the entrapment efficiency (EE %) and drug
loading (DL %) of insulin and the HA2 peptide,19 SLNs were
dispersed in pH 2 HCl solutions and centrifuged at 13 000 rpm for 30
min. The amounts of insulin and HA2 peptide in supernatants were
quantified by a reverse-phase high performance liquid chromatography
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(HPLC) (Agilent 1200 series and Alltech, respectively) method, and
free FITC-INS was quantified by a multimode reader.10
2.3. Colloidal Stability of SLNs. The colloidal stability of the
SLNs was investigated by incubating SLNs with simulated gastric fluid
[SGF, with and without pepsin (0.32%, w/v)] and simulated intestinal
fluid [SIF, with and without trypsin (1%, w/v)] at 37 °C.23
Furthermore, SGF was preneutralized by sodium bicarbonate solution
(3%, w/v).24,25 At predetermined time intervals, the particle size of
SLNs was determined. TEM was also used to confirm the results.
2.4. Drug and HA2 Peptide Release of SLNs. The in vitro
release of insulin and HA2 peptide from SLNs was investigated using
the dialysis method.26−28 Briefly, 2 mL of SLNs was added in dialysis
tubing (MWCO: 100 kDa) and then dialyzed against 1000 mL of
release medium (pH 2, 5.5, and 6.8) at 37 °C. At a predetermined
time, the concentrations of insulin and HA2 peptide in dialysis tubing
were determined by RP-HPLC. The in vitro release in the pH 2
medium neutralized by sodium bicarbonate solution was also
performed.
2.5. Cell Experiments. 2.5.1. Cell Culture and Cytotoxicity
Assay. Human colon carcinoma Caco-2 cells were obtained from the
Institute of Biochemistry and Cell Biology (Shanghai, China) and
cultured as the method described before.10,29 The in vitro cytotoxicity
of SLNs was evaluated. In brief, the Caco-2 cells were incubated with
SLNs for 3 h and were applied to methylthiazolyl tetrazolium (MTT)
assay.10 Hank’s balanced salt solution (HBSS)-treated group was used
as the control group.
2.5.2. In Vitro Cellular Uptake. 2.5.2.1. Flow Cytometry Method.
The cellular uptake of SLNs was quantified by flow cytometry.30 In
brief, Caco-2 cells were seeded in 12-well plates for 5 days. Then,
Caco-2 cells were incubated with fluorescence-labeled SLNs (FITC-
insulin, 100 μg/mL) at 37 °C for 3 h. Afterward, the cells were
trypsinized and redispersed in cold phosphate-buffered saline (PBS)
and then analyzed by flow cytometry analysis (CYTOMICS FC500,
Beckman Coulter, Miami, FL, USA).
2.5.2.2. Endocytic Mechanism of SLNs. To investigate the
endocytic mechanism of SLNs, the inhibitors of metabolism (5 mM
sodium azide), clathrin-mediated endocytosis (30 μM chlorproma-
zine), caveolae-mediated endocytosis (500 nM filipin), and micro-
pinocytosis (12 μg/mL amiloride) were incubated with Caco-2 cells at
37 °C for 30 min.31,32 Afterward, the cells were incubated with SLNs
and the same amount of inhibitors for 3 h. The internalization was
analyzed by flow cytometry.
2.5.2.3. Enzyme-Linked Immunosorbent Assay (ELISA) Method.
The ELISA method was selected to test the internalization of insulin
with activity in cells. Caco-2 cells were incubated with various SLN
suspensions (insulin, 100 μg/mL) at 37 °C for 3 h. Then, the cell
extracts containing active insulin were obtained by several freeze−thaw
cycles. The concentration of active insulin in supernatants of cell
extracts was measured by using the porcine insulin ELISA kit (R&D
System, Inc., MN, USA)33 and calibrated by the BCA assay kit
(KeyGen Biotech Co., Ltd., Nanjing, China).
2.5.3. Intracellular Endo−Lysosomal Trafficking of SLNs.
2.5.3.1. Colocalization of SLNs in Endosomes and Lysosomes.
The localization of SLNs with different endosomes (early endosomes
and late endosomes) was analyzed using immunofluorescence
staining.10 Briefly, the cells were then incubated with SLNs at 37 °C
for 1 h. After incubation time, the cells were rinsed by PBS, fixed with
4% paraformaldehyde for 15 min, permeabilized by 1% Triton X-100
for 10 min, and blocked with 5% normal goat serum for 1 h. Then,
primary rabbit anti-Rab5 and mouse anti-Rab7 antibodies were added
separately at 4 °C overnight. The secondary antibodies 594-labeled
goat anti-rabbit and anti-mouse IgG were incubated with the cells at 37
°C for 2 h. Additionally, to observe the localization of SLNs with
lysosomes, Caco-2 cells were incubated with 50 nM LysoTracker
probe at 37 °C for 30 min. DAPI was used to stain cell nuclei before
CLSM observation.10
2.5.3.2. Hemolysis Assay. To determine the pH-sensitive
amphiphilic endosomal escape of various SLNs, hemolytic assay was
used as previous studies.34,35 Briefly, red blood cells (RBCs) obtained
from Sprague-Dawley (SD) rats were diluted 1:50 in PBS at pH 7.4,
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Figure 1. (A) Schematic diagram of SLNs. (B) Size distribution of INS SLNs (a), INS HA2-W-SLNs (b), and INS HA2-O-SLNs (c) performed by
Malvern NanoSight LM10 system. (C) TEM images of INS SLNs (a), INS HA2-W-SLNs (b), and INS HA2-O-SLNs (c). Scale bar: 100 nm.
6.5, and 5.5. Then, INS SLNs, INS HA2-W-SLNs, INS HA2-O-SLNs,
HA2 (HA2, 30 μg/mL), and Triton X-100 (1%, w/v) were incubated
with the RBC solutions at 37 °C for 2 h. A multimode reader was
applied to detect the absorbance of the supernatant at 540 nm. 1%
Triton X-100 was used as the reference for 100% hemolytic activity.
2.5.4. Transepithelial Transport Study. The transepithelial trans-
port study of the tested samples was investigated on the Caco-2
monolayers seeded on Transwell inserts.36 Cell monolayers were first
equilibrated at 37 °C by HBSS. After equilibration, the cell monolayers
were incubated with INS SLNs, INS HA2-W-SLNs, INS HA2-O-
SLNs, and free insulin (insulin, 100 μg/mL) for 6 h at 37 °C. The
amount of permeated insulin in the basolateral chambers was
measured by the ELISA kit. The transepithelial electrical resistant
(TEER) values were measured before and after incubation to explore
the integrity of cell monolayers.
2.6. Ex Vivo Ligated Intestinal Loop Assay. The permeation of
insulin in the intestinal loop was evaluated by ex vivo ligated intestinal
loop assay.37 The animal study protocol was approved by the
Institutional Animal Care and Use Committee of Sichuan University.
Male SD rats (220−250 g) were sacrificed. Then, 2 cm sections of the
duodenum, jejunum, and ileum were obtained, carefully washed, and
ligated at both ends. Then, the suspensions of SLNs (pH 5.5 or pH
6.8) were injected into the loops. In addition, the sections were
incubated with pH 7.4 medium at 37 °C for 2 h. The amount of
permeated insulin in the medium was determined by the ELISA kit.
2.7. Pharmacological and Pharmacokinetics Studies. The
pharmacological and pharmacokinetics studies of various SLNs were
performed on diabetic rats.38 The male SD rats (220−250 g) were
injected by streptozotocin (70 mg/kg) solution to induce diabetes.
Rats with the fasting blood glucose above 16 mM were diabetic
models. Prior to the experiments, rats were fasted overnight with the
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supplement of water. Before oral administration of SLNs, 0.5 mL of
sodium bicarbonate solution (3%, w/v) was orally administrated to
neutralize the gastric acid. Then, free insulin solution (50 IU/kg), INS
SLNs (50 IU/kg), INS HA2-W-SLNs (50 IU/Kg ) and INS HA2-O-
SLNs (50 IU/kg) were intragastrically administrated. Also, insulin
solution (5 IU/kg) was subcutaneously administrated. At different
time intervals, blood glucose levels of rats were determined by a
glucose meter. Meanwhile, plasma insulin levels were measured by the
ELISA kit. The pharmacological availability (PA %) and relative
bioavailability (FR %) to subcutaneous injection were calculated using
the following formulas:
AACoral × doses.c.
PA (%) = × 100
AACs.c. × doseoral
AUCoral × doses.c.
FR (%) = × 100
AUCs.c. × doseoral
2.8. Statistical Analyses. All experiments were performed in
triplicate unless otherwise stated and presented as mean ± standard
deviation. Statistical analyses of the data were conducted with SPSS
program 16.0 and two-tail Student’s t-test. Statistical significance was
defined when p < 0.05.
3. RESULTS AND DISCUSSION
3.1. Preparation and Characterization of SLNs. SLNs
were prepared by the W/O/W double emulsion strategy, which
has been commonly used to load hydrophobic and hydrophilic
macromolecules.18,39 The primary W/O emulsion was first
obtained, and the emulsion was then emulsified in a continuous
aqueous phase to form the double emulsion, which contained
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Table 1. Characterization of Various Insulin-Loaded SLNs and FITC-Insulin-Loaded SLNs Prepared by the Optimized
Formulationa
samples size (nm) PDI zeta (mV) EEinsulin (%) EEHA2 (%) DL (%)
INS SLN 171.2 ± 1.6 0.23 ± 0.02 −9.2 ± 0.2 97.93 ± 0.13 8.03 ± 0.19
INS HA2-W-SLN 148.8 ± 4.2 0.17 ± 0.02 −13.1 ± 1.3 98.90 ± 0.35 75.97 ± 0.65 7.23 ± 0.26
INS HA2-O-SLN 161.6 ± 3.7 0.25 ± 0.03 −16.1 ± 0.5 98.16 ± 0.80 92.67 ± 1.11 7.52 ± 0.31
FITC-INS SLN 172.4 ± 1.5 0.22 ± 0.03 −8.4 ± 1.1 96.60 ± 0.33 7.91 ± 0.15
FITC-INS HA2-W-SLN 157.8 ± 3.1 0.24 ± 0.02 −11.0 ± 0.3 97.64 ± 0.15 78.64 ± 0.91 7.42 ± 0.26
FITC-INS HA2-O-SLN 160.2 ± 2.0 0.26 ± 0.03 −14.6 ± 0.9 97.60 ± 0.26 90.83 ± 0.76 7.71 ± 0.24
a
Data shown as mean ± SD (n = 3).

Figure 2. Variation in the particle size of insulin-loaded SLNs following incubation in (A) SGF with 0.32% pepsin, (B) neutralized SGF with 0.32%
pepsin, (C) SIF with 1% trypsin at predetermined time intervals (mean ± SD, n = 3). *p < 0.05 vs INS SLNs at 0 h; #p < 0.05 vs INS HA2-W-SLNs
at 0 h; &p < 0.05 vs INS HA2-O-SLNs at 0 h. (D) TEM images of INS HA2-O-SLNs (a), INS HA2-O-SLNs in SGF with 0.32% pepsin (b), INS
HA2-O-SLNs in neutralized SGF with 0.32% pepsin (c), and INS HA2-O-SLNs in SIF with 1% trypsin (d). Scale bar: 100 nm.

insulin in the inner aqueous phase, lipid matrix in the oil phase,
and Pluronic F68 as the emulsifier in the outer aqueous phase.
Finally, the evaporation of the solvent from the W/O/W
emulsion allowed the formation of SLNs.40 The combined use
of soybean lecithin, tripalmitin, and stearic acid has been
previously proven to increase the crystallinity of NPs, which
could prevent the leakage of the incorporated insulin (Figure
S1).18 Meanwhile, sodium cholate,41 an amphipathic bio-
surfactant, was introduced to encapsulate hydrophilic insulin in
the internal aqueous phase to further improve the entrapment
efficiency and provide the sufficient protection for drugs via the
hydrophobic effect between the amphipathic biosurfactant and
peptides.18,42
The formulation and preparation processes were optimized
(Table S1). HA2 peptide-containing SLNs were prepared by
loading HA2 peptide in the internal aqueous phase or oil phase
(INS HA2-W-SLNs or INS HA2-O-SLNs) (Figure 1A). For
preparation of INS HA2-O-SLNs, a mixed organic solvent was
used to increase the solubility of HA2 peptide, achieving the
successful entrapment of HA2 peptide mainly in the outer lipid
shell. The characterizations of the obtained SLNs were
presented in Table 1. Small PDI index (under 0.3) indicated
the stability of various SLNs in terms of narrow size
9318
distribution. Also, the mean particle sizes of the formulated
SLNs were in a range of 150 nm to 170 nm (Table 1, Figure
1B). Excitingly, the optimal SLNs exhibited high insulin
entrapment efficiency (over 97%) and drug loading efficiency
(over 7%), indicating that introducing appropriate noncovalent
interactions (hydrophobic effect, hydrogen bonding, π-effects,
etc.) and preparation methods could efficiently solve the
contradiction between the hydrophobic lipid matrix and
hydrophilic proteins.42−44
Meanwhile, both INS HA2-W-SLNs and INS HA2-O-SLNs
showed high HA2 peptide entrapment efficiency (over 75%),
demonstrating the capacity of the vehicles encapsulating both
hydrophobic and hydrophilic agents. Insulin-loaded SLNs (INS
SLNs) were negatively surface charged (−9.2 mV), while
insulin and HA2 peptide dual-loaded SLNs (INS HA2-O-SLNs
and INS HA2-W-SLNs) showed even more negative surface
charge (−16.1 and −13.1 mV) because of the existence of the
anionic HA2 peptide.45 The different surface charges between
INS HA2-O-SLNs and INS HA2-W-SLNs suggested that the
HA2 peptide was mainly installed in different layers of the
particles. In addition, the confocal images of TRITC-insulin
and FITC-HA2 peptide dual-loaded SLNs also confirmed the
results (Figure S2). Moreover, the preparation technique
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Figure 3. Accumulation release profiles of insulin against PBS at (A) pH 5.5 and (B) pH 6.8 (mean ± SD, n = 3). *p < 0.05 vs INS HA2-W-SLNs of
INS HA2 SLNs; #p < 0.05 vs INS HA2-W-SLNs of INS HA2-O-SLNs. Accumulation release profiles of HA2 peptide against PBS at (C) pH 5.5 and
(D) pH 6.8 (mean ± SD, n = 3). ^p < 0.05 vs INS HA2-W-SLNs of INS HA2-O-SLNs.
avoided the high cost of grafting HA2 peptide either with
drugs46 or nanocarriers.47 The characterizations of the obtained
FITC-insulin-loaded SLNs are shown in Table 1, which were
similar to those of insulin-loaded SLNs. The TEM images
showed that the morphology of all SLNs was spherical in shape
(Figure 1C).
3.2. Colloidal Stability of SLNs. For orally delivered NPs,
it is of importance to overcome the harsh environment of the
GIT before NPs intrude the epithelia cells. Thus, the evaluation
of the colloidal stability of SLNs in simulated GI fluid (SGF and
SIF) is essential. The aggregation of SLNs was observed in
SGF, whereas SLNs remained unchanged in neutralized SGF
(Figures 2A,B and S3A,B). Various SLNs presented no
significant aggregation or disintegration in SIF with or without
trypsin (1%, w/v) (Figures 2C and S3C). Additionally, regular
and smooth morphology of particles was observed when SLNs
were dispersed in enzyme-containing neutralized SGF and SIF
(Figure 2D), suggesting that the colloidal stability of SLNs
would not be affected by the main proteases in the GIT.
However, SLNs in SGF could be observed by aggregation and
morphological changes (Figure 2D(b)). In the present study,
before oral administration of SLNs, sodium bicarbonate
solution was intragastrically administrated to neutralize the
gastric acid following previous reports.24,25 In clinical setting,
enteric-coated gelatin capsules or tablets could also be utilized
to prevent the exposure of SLNs in acidic gastric fluids.
3.3. Drug and HA2 Peptide Release of SLNs. The in
vitro release profiles of insulin and HA2 peptide in pH 2 buffer
are shown in Figure S4. Approximately 40% insulin and 65%
HA2 peptide released from INS HA2-O-SLNs within 2 h.
While in the neutralized medium, both HA2 peptide and
insulin showed sustained release from SLNs. Moreover, the
release rate of HA2 peptide from INS HA2-W-SLNs was
significantly lower than that from INS HA2-O-SLNs.
Then, we studied insulin and HA2 peptide release in pH 5.5
and 6.8 media, which mimicked the pH environments in the
acidic endosomes48 and small intestine,18 respectively. INS
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HA2-O-SLNs and INS HA2-W-SLNs showed different trends
of release for insulin and HA2 peptide. The release profile of
insulin from INS HA2-W-SLNs was more sustained compared
with that of INS HA2-O-SLNs and INS SLNs. After incubation
for 12 h, approximately 55 and 35% encapsulated insulin were
released from the INS HA2-W-SLNs in the pH 6.8 and 5.5
buffer, respectively, which were significantly lower compared
with that from the INS SLNs and INS HA2-O-SLNs (∼70 and
∼50%, p < 0.05) (Figure 3A,B). Interestingly, the release at pH
5.5 was lower than that at pH 6.8 for all of the samples. As
insulin (isoelectric point = 5.3) in media at pH 5.5 was
dominantly in nonionic form, the solubility at pH 5.5 was much
lower than that at pH 6.8. Therefore, the decrease of the release
rate at pH 5.5 might be due to the low solubility of insulin.49,50
Additionally, a much faster release rate of HA2 peptide
located in the external oil phase (INS HA2-O-SLNs) was also
observed compared with that of HA2 peptide located in the
internal aqueous phase (INS HA2-W-SLNs) at pH 5.5 (Figure
3C), which was similar to the insulin release because of the
interaction of HA2 peptide and insulin in the aqueous phase. A
similar phenomenon was also observed in pH 6.8 (Figure 3D).
This result demonstrated that the spatial location for HA2
peptide could greatly influence the release rate. Therefore, with
a much faster release rate of HA2 peptide loaded in the external
oil phase, instead of the internal aqueous phase, INS HA2-O-
SLNs were expected to acquire a desirable ability of rapid
endosomal escape.
3.4. Cell Experiments. 3.4.1. Caco-2 Cell Viability Study.
Caco-2 cells were used as the cell model for the cell
experiments because of the advantage of perfectly mimicking
the enterocytes of the intestinal gut. The biocompatibility of
SLNs was evaluated by MTT assay. As the viability of both
insulin- and FITC-labeled insulin encapsulated in SLNs was
ranged from 80 to 120%, all SLNs at test concentrations
exhibited no cytotoxicity (Figure S5).
3.4.2. Cellular Uptake. To investigate whether the addition
of HA2 peptide could affect the cellular uptake of SLNs, flow
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Figure 4. (A) Flow cytometry quantification of internalization of FITC-labeled INS SLNs, INS HA2-W-SLNs, and INS HA2-O-SLNs (mean ± SD,
n = 3). (B) Endocytic mechanism of SLNs in terms of energy dependence, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and
micropinocytosis. *p < 0.05 vs control group of INS SLNs; &p < 0.05 vs control group of INS HA2-W-SLNs; #p < 0.05 vs control group of INS
HA2-O-SLNs. (C) ELISA quantification of INS SLNs, INS HA2-W-SLNs, and INS HA2-O-SLNs on Caco-2 cells at tested insulin concentration
(mean ± SD, n = 3). **p < 0.01 vs INS HA2-O-SLNs. (D) CLSM images of SLNs (green) and Caco-2 cells stained with early endosomes (red) and
late endosomes (red). (E) CLSM images of SLNs (green) and Caco-2 cells stained with lysosomes (red). Yellow color denotes the overlay of
colocalization of red and green fluorescence. DAPI staining of nuclei (blue).

cytometry was utilized to quantify the amount of fluorescence-
labeled SLNs internalized into cells. Interestingly, as shown in
Figure 4A, the detected fluorescent intensity showed no
significant difference among all samples, indicating that the
endocytosis was not influenced by the addition of HA2 peptide.
Furthermore, the endocytic mechanism of various SLNs was
evaluated. All tested formulations exhibited obvious reduction
of cellular uptake in the presence of sodium azide,
chlorpromazine, and amiloride (Figure 4B), suggesting that
clathrin-mediated endocytosis was the endocytic mechanism of
SLNs involved.
Meanwhile, the retained active insulin after cellular internal-
ization was determined by ELISA assay. The activity of proteins
with fragile structures was easily disrupted under harsh
conditions.51,52 During the transport processes, the internalized
insulin might lose biological activity because of the complex
environment within cells.33 The ELISA method used in our
study proved that insulin contained in all samples could retain
partly biological activity after cellular internalization. More
excitingly, INS HA2-O-SLNs displayed the greatest insulin
activity (2.64 folds, p < 0.05) compared with INS SLNs and
INS HA2-W-SLNs, which exhibited similar amounts of active
insulin in cells (Figure 4C). These results indicated that HA2
peptide with the appropriate application played a crucial role in
protecting the biological activity of insulin though it did not
elevate the uptake of SLNs.
9320
3.4.3. Endosomal Escape of SLNs. SLNs have been
confirmed to involve in clathrin-mediated endocytosis. After
the sequential transport through early and late endosomes,
SLNs transported through an intracellular endolysosomal
pathway will enable the biotherapeutics delivered to unique
enzymatic degradative compartments named lysosomes.53 Rab
GTPases play a vital role on vesicular transport in the
endolysosomal pathway.54 Rab5 and Rab7 are expressed in
early endosomes and late endosomes, respectively.55,56 As
expected, the colocalization of FITC-labeled INS SLNs, INS
HA2-W-SLNs, and INS HA2-O-SLNs with early endosome
compartments (Rab5) was observed (Figure 4D), suggesting
that all formulations were involved in early endosomes. It was
reported that after endocytosis, most NPs would first be
transported to early endosomes, and then, they could be
trafficked to other organelles.57,58 Interestingly, INS HA2-O-
SLNs, showing faster release of HA2 peptide, displayed less
colocalization with late endosomes and lysosomes. While INS
SLNs and INS HA2-W-SLNs significantly overlaid with the late
endosomes and lysosomes. Thus, the results indicated the
crucial role of HA2 peptide on the endosomal escape of cargoes
by destabilizing the membrane of endosomes. For INS HA2-W-
SLNs, however, HA2 peptide and insulin would be exposed in
late endosomes or lysosomes simultaneously after degradation
of the lipid materials,19,59 resulting in the entrapment and
degradation of drugs in lysosomes. In accordance with the in
DOI: 10.1021/acsami.8b00507
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ACS Applied Materials & Interfaces Research Article

Figure 5. (A) Relative hemolysis efficiency of 1% triton X-100, various insulin loaded SLNs and HA2 peptide at pH 7.4, 6.5, and 5.5, and PBS as the
control group. *p < 0.05 vs pH 7.4; #p < 0.05 vs pH 6.5. (B) Transepithelial transport of insulin and insulin-loaded SLNs. *p < 0.05 vs INS; #p < 0.05
vs INS SLNs; &p < 0.05 vs INS HA2-W-SLNs. (C) TEER value of Caco-2 cell monolayers before and after incubation of insulin or SLNs. (D)
Permeated amount of transported insulin in ligated intestinal loop assay. *p < 0.05 vs INS SLNs; #p < 0.05 vs INS HA2-W-SLNs; &p < 0.05 vs INS
HA2-O-SLNs in duodenum at pH 6.8.

vitro cellular uptake assays, INS HA2-O-SLNs demonstrated
the highest efficiency of endosomal escape by the interactions
between HA2 and the membrane of late endosomes, protecting
insulin from the transport to lysosomes.
Meanwhile, the mechanism of endosomal escape of SLNs
was further investigated using hemolytic assay.34,35 It was
demonstrated that the relative hemolytic activity of INS HA2-
O-SLNs increased with the acidification of maturing endo-
somes (Figure 5A). The hemolysis of INS HA2-O-SLNs is
15.53 and 52.61% at pH 6.5 and 5.5, respectively, suggesting
the excellent ability of INS HA2-O-SLNs to interact with late
endosomes; while, INS HA2-O-SLNs exhibited low hemolytic
activity (2.85%) at pH 7.4, indicating the low toxicity on cells.
In contrast, INS HA2-W-SLNs presented relative low pH-
sensitive amphiphilic endosomal escape. The hemolytic activity
of INS SLNs did not increase with the acidification of
endosomes. This result was consistent with the intracellular
trafficking of various SLNs observed under CLSM, and INS
HA2-O-SLNs were confirmed to display the pH-sensitive
amphiphilic endosomal escape.
3.4.4. Transepithelial Transport Study. INS HA2-O-SLNs
have been confirmed to protect insulin from lysosomal
degradation by successful endosomal escape. The permeation
of insulin through Caco-2 cell monolayers was further
investigated. As shown in Figure 5B, after loaded into SLNs,
insulin showed the enhanced transepithelial transport efficiency
(p < 0.05). Of note, insulin of INS HA2-O-SLNs exhibited the
highest accumulated amount in the basolateral side, which was
respectively 4.30-fold (p < 0.05) and 2.19-fold (p < 0.05)
higher than that of free insulin and INS SLNs. By contrast,
9321
insulin of INS HA2-W-SLNs merely presented a slight increase
of transepithelial transport efficiency than that of free insulin
and INS SLNs (p < 0.05). Thus, INS HA2-O-SLNs were
confirmed to promote the transepithelial transport, which
might be due to the protection of insulin during the
intracellular trafficking. Meanwhile, the TEER value remained
unchanged after incubation of various SLNs (Figure 5C),
indicating that the monolayer remained intact during the
transcellular transport of insulin.
3.5. Ex Vivo Ligated Intestinal Loop Assay. The oral
intestinal absorption of INS HA2-O-SLNs was further
investigated by the ex vivo ligated intestinal loop assay (Figure
5D).37 Consistent with the in vitro behavior of SLNs, INS
HA2-O-SLNs showed 1.48 fold higher intestinal transport than
INS SLNs and INS HA2-W-SLNs in jejunum. Interestingly, the
absorption of SLNs in jejunum was much higher than that in
duodenum and ileum, which might be due to the more dense
jejunum villi distribution in the small intestine.60 Moreover,
INS HA2-O-SLNs dispersed in pH 5.5 enhanced insulin
transport through the duodenum, while no significant increase
was observed in INS SLNs or INS HA2-W-SLNs in the same
condition. The result suggested that INS HA2-O-SLNs could
potentially enhance the oral absorption of insulin in duodenum.
However, SLNs might not have enough retention time in
duodenum on animal models. The enhanced duodenal
absorption of INS HA2-O-SLNs may be weakened by intestinal
flow, rapid dilution, as well as spreading.61
3.6. Pharmacological and Pharmacokinetics Studies.
The in vivo pharmacological and pharmacokinetics effect of
various SLNs were determined on diabetic rats. As shown in
DOI: 10.1021/acsami.8b00507
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ACS Applied Materials & Interfaces Research Article

Figure 6. (A) Blood glucose level (% of initial) and (B) serum insulin level of rats following administration of various formulations (mean ± SD, n =
5). *p < 0.05 vs insulin solution. #p < 0.05 vs INS SLNs. &p < 0.05 vs INS HA2-W-SLNs.

Figure 6A, subcutaneously injected insulin (5 IU/kg) lipid shell containing endosomal escape agent (HA2 peptides)
represented remarkable hypoglycemia effect and the blood and an aqueous core containing insulin. HA2 peptide in the
glucose level decreased to the minimum at 2 h, whereas oral external oil phase (INS HA2-O-SLNs) was demonstrated to
administration of insulin (50 IU/kg) failed to reduce the blood possess a much faster release compared with that of HA2 in the
glucose level. Oral administration of INS SLNs, INS HA2-W- external aqueous phase (INS HA2-W-SLNs). When exposed to
SLNs, and INS HA2-O-SLNs generated significant hypoglyce- acidic endosomes, the released HA2 peptide helped cargoes
mic response (p < 0.05), indicating that bioactivity of insulin escape from degradation in the lysosomal pathway by rapidly
encapsulated in SLNs was protected against various pH disrupting the late endosomal membranes. In vitro cell
environments and enzymatic digestion. Excitingly, INS HA2- experiments confirmed that INS HA2-O-SLNs could retain
O-SLNs presented the most excellent hypoglycemic effect with the highest biological activity of internalized insulin. As
37% blood glucose decrease at 3 h. The pharmacological demonstrated in the intracellular trafficking of SLNs, INS
availability (PA %) of different samples related to subcutaneous HA2-O-SLNs displayed much less distribution in late endo-
injection is shown in Table 2. INS HA2-O-SLNs demonstrated somes and lysosomes. Meanwhile, INS HA2-O-SLNs exhibited
PA % of 7.32%, which was 4 and 1.7 folds higher than free the highest transepithelial transport efficiency, with 2.19 folds
insulin solution and INS HA2-W-SLNs. (p < 0.05) and 1.72 folds (p < 0.05) higher intestinal transport
as compared to INS SLNs and INS HA2-W-SLNs.
Table 2. Pharmacological and Pharmacokinetic Parameters Furthermore, the ex vivo ligated intestinal loop assay
of Insulin Formulations in a Diabetic Rat Modela demonstrated that insulin from INS HA2-O-SLNs exhibited
the highest permeation in various regions of intestines. Finally,
samples dose (IU/kg) AUC (mIU·h/L) PA (%) F (%) the developed SLNs were confirmed to increase the serum
SC insulin 5 278.1 ± 69.2 100 insulin concentration and generate an excellent hypoglycemic
oral insulin 50 47.3 ± 6.1 1.83 1.70 response, suggesting the importance of endosomal escape for
INS SLN 50 96.5 ± 10.5 3.40 3.47 orally delivered proteins and peptides. The excellent in vitro−in
INS HA2-W-SLN 50 96.6 ± 23.2 4.33 3.47 vivo correlation confirmed the great potency for SLNs with
INS HA2-O-SLN 50 152.1 ± 12.0 7.32 5.47 endosomal escape function, and the strategy could be further
a
AUC: area under the plasma concentration−time curve; PA %: applied in the oral delivery of other biomacromolecules.

■
pharmacological bioavailability; F %: relative bioavailability.
ASSOCIATED CONTENT
The serum insulin concentration is shown in Figure 6B. *
S Supporting Information
Compared with subcutaneously administrated free insulin, SLN The Supporting Information is available free of charge on the
administration by gavage showed a relatively slower increase in ACS Publications website at DOI: 10.1021/acsami.8b00507.
the serum insulin concentration. At the dose of 50 IU/kg, INS The chemical structure of glycerol tripalmitate and
HA2-O-SLNs showed a significant higher relative bioavailability stearic acid; Physicochemical characterization of SLNs;
of 5.47%, which was 3.2-fold higher than free insulin.
CLSM images of HA2-O-SLNs and HA2-W-SLNs;
Therefore, the in vivo−in vitro correlation was confirmed.
accumulation release profiles of insulin and HA2 in pH
Taken previous results together, HA2 peptide in INS HA2-O-
SLNs could be appropriately released and destabilize the 2; and Caco-2 cell viability treated with various SLNs by
membrane of endosomes to achieve the endosomal escape of MTT assays (PDF)
insulin. The designed delivery system showed the great potency
as a promising strategy to overcome degradation of insulin in
lysosomes and further enhanced the oral bioavailability of
■ AUTHOR INFORMATION
Corresponding Author
insulin.
*E-mail: huangyuan0@163.com. Phone/Fax: +86-28-
4. CONCLUSIONS 85501617.
In summary, we rationally developed the functional nano- ORCID
vehicles for the efficient oral absorption of biomacromolecules. Zhirong Zhang: 0000-0001-9118-5394
The optimized SLNs (INS HA2-O-SLNs) were comprised of a Yuan Huang: 0000-0003-3410-8602
9322 DOI: 10.1021/acsami.8b00507
ACS Appl. Mater. Interfaces 2018, 10, 9315−9324
ACS Applied Materials & Interfaces
Author Contributions
†
Y.X. and Y.Z. contributed equally to this study and shared first
authorship.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors gratefully acknowledge financial support from the
National Science Foundation for Distinguished Young Scholars
(81625023) and the Major Research Plan of National Natural
Science Foundation of China (81690261).
■
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9324 DOI: 10.1021/acsami.8b00507

ACS Appl. Mater. Interfaces 2018, 10, 9315−9324