Glucose-Responsive Supramolecular Vesicles Based on Water-Soluble Pillar[5]arene and Pyridylboronic Acid Derivatives for Controlled Insulin Delivery

Article abstract of DOI:10.1002/chem.201700345

The stimuli-responsive behavior of supramolecular nanocarriers is crucial for their potential applications as smart drug delivery systems. We hereby constructed a glucose-responsive supramolecular drug delivery system based on the host–guest interaction between a water-soluble pillar[5]arene (WP5) and a pyridylboronic acid derivative (G) for insulin delivery and controlled release under physiological conditions. The approach represents the ideal treatment of diabetes mellitus. The drug loading and in vitro drug release experiments demonstrated that large molecular weight insulin could be encapsulated into the vesicles with high loading efficiency, which, to our knowledge, is the first example of small-size supramolecular vesicles with excellent encapsulation capacity of a large protein molecule. Moreover, FITC-labeled insulin was used to evaluate the release behavior of insulin, and it was demonstrated that high glucose concentration could facilitate the quick release of insulin, suggesting a smart drug delivery system for potential application in controlled insulin release only under hyperglycemic conditions. Finally, we demonstrated that these supramolecular nanocarriers have good cytocompatibility, which is essential for their further biomedical applications. The present study provides a novel strategy for the construction of glucose-responsive smart supramolecular drug delivery systems, which has potential applications for the treatment of diabetes mellitus.

Full text of DOI:10.1002/chem.201700345

A Journal of
Accepted Article
Title: Glucose-Responsive Supramolecular Vesicles Based on Water-
Soluble Pillar[5]arene and Pyridylboronic Acid Derivative for
Controlled Insulin Delivery
Authors: Lei Gao, Tingting Wang, Keke Jia, Xuan Wu, Chenhao Yao,
Wei Shao, Dongmei Zhang, Xiao-Yu Hu, and Leyong Wang
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To be cited as: Chem. Eur. J. 10.1002/chem.201700345
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Glucose-Responsive Supramolecular Vesicles Based on Water-
Soluble Pillar[5]arene and Pyridylboronic Acid Derivative for
Controlled Insulin Delivery
Lei Gao,[a] Tingting Wang,[a] Keke Jia,[b] Xuan Wu,[a] Chenhao Yao,[a] Wei Shao,[a] Dongmei Zhang,[b]
Xiao-Yu Hu,*[a] and Leyong Wang *[a,c]
Abstract: The stimuli-responsive behavior of supramolecular
nanocarriers is crucial for their potential applications as smart drug
delivery systems. We hereby constructed a glucose-responsive
supramolecular drug delivery system constructed by the host−guest
interaction between a water-soluble pillar[5]arene (WP5) and a
pyridylboronic acid derivative (G) for insulin delivery and controlled
release at physiological condition, which represents the ideal
treatment of diabetes mellitus. The drug loading and in vitro drug
release experiments demonstrated that large molecular weight
insulin could be successfully encapsulated into the vesicles with high
loading efficiency, which to the best of our knowledge, is the first
example of small size supramolecular vesicles with excellent
encapsulation capacity of large protein molecule. Moreover, FITC-
labeled insulin was used to evaluate the release behavior of insulin,
and it was demonstrated that high glucose concentration could
facilitate the quick release of insulin, suggesting a smart DDS for the
potential application in controlled insulin release only under
hyperglycemic conditions. Finally, we demonstrated that these
supramolecular nanocarriers have good cytocompatibility, which is
essential for their further biomedical applications. The present study
provides a novel strategy for the construction of glucose-responsive
smart supramolecular drug delivery system, which has potential
applications for the treatment of diabetes mellitus.
injections of insulin and regular monitoring of the blood glucose
levels after diets, which have brought great agony and
inconvenience to the patients. More importantly, for conventional
treatment, the glucose sensing and drug therapy are not directly
coupled, which does not tightly regulate glucose levels in
patients. Therefore, lack of tight control of blood glucose levels
is often associated with significant side-effects including fatal
hypoglycemia, blindness, and kidney failure.[2] In order to
improve the living quality of diabetic patients, it is essential and
important to design and develop efficient and smart drug
delivery systems (DDSs) with glucose-responsive function,
which can only release insulin in response to the hyperglycemic
conditions.
Smart drug delivery systems or drug nanocarriers refer to the
DDSs with responsive behavior to the external stimuli such as
pH, [3] temperature, [4] light, [3b, 5] enzyme, [6] and chemo-stimuli, [7]
which can not only delivery drugs to the target disease sites, but
also can release them in response to the changes in external
environment, especially under the specific microenvironment of
some organs, intracellular space, or pathological sites.[8] In
recent years, with the rapid development of supramolecular
chemistry, supramolecular amphiphiles that constructed based
on the non-covalent interactions, especially host−guest
interactions between macrocyclic hosts and guest molecules
have been extensively employed to constructed smart
nanocarriers. Moreover, due to the dynamic nature of non-
covalent interactions, supramolecular amphiphiles have the
special ability to undergo reversible switching of structures,
morphologies, and functions in response to certain external
stimuli.[9] which have great potential applications for controlled
drug delivery and release. So far, inspired by the excellent
binding reaction between boric acid and cis-diols, glucose-
responsive materials have also been well-developed, but there
are only a few reports on the construction of functional drug
delivery systems for the controlled delivery of insulin.[10]
Moreover, among various reported glucose-responsive DDSs,
most of them were focused on the construction of mesoporous
silica nanoparticles or polymeric materials.[10a, 11] However, very
few research work has been conducted on the construction of
glucose-responsive supramolecular DDSs based on supra-
amphiphiles, which could endow the obtained nanoparticles with
excellent smart behaviors.
Introduction
Diabetes mellitus, as a kind of metabolic disorder disease, is
characterized by accumulating concentration of glucose in
plasma due to the insufficient secretion of insulin from
pancreas.[1] Currently, the main treatment for diabetes is daily
[a]
L. Gao, T. Wang, Dr. X.-Y. Hu,* Prof. Dr. L. Wang*
Key Laboratory of Mesoscopic Chemistry of MOE, Collaborative
Innovation Center of Chemistry for Life Sciences, School of
Chemistry and Chemical Engineering
Nanjing University
Nanjing 210023 (China)
E-mail: lywang@nju.edu.cn (LW); huxy@nju.edu.cn (XH)
K. Jia, Dr. D. Zhang,
State Key Laboratory of Pharmaceutical Biotechnology, School of
Life Sciences
Nanjing University
Nanjing 210023 (China)
[b]
[c]
Recently, research on pillararene-based supramolecular
materials has received burgeoning attention and advanced
Prof. Dr. L. Wang*
rapidly.[12] Our group has also developed
a
series of
Institute for Natural & Synthetic Organic Chemistry
Changzhou University
Changzhou 213164 (China)
supramolecular amphiphiles based on water-soluble pillararenes,
which can further assemble to form higher-ordered aggregates
such as vesicles, micelles, and nanoparticles to achieve
13]
controllable drug delivery.[12f-j,
On the basis of the above
Supporting information for this article is given via a link at the end of
the document.
researches, it could be concluded that water-soluble pillararene
contains a hydrophobic cavity and hydrophilic terminal groups
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distributed on both rims of the macrocycle should be an ideal
candidate for the construction of supramolecular amphiphiles
due to their symmetrical pillar and unique rigid architectures,
excellent properties in host−guest chemistry, as well as
outstanding biocompatibility.[12a, 13-14] On the other hand, it is well
known that heterocyclic-containing boric acid derivatives can
bind D-glucose under approximate neutral pH range due to the
presence of electron-rich hetero atom such as N, O or S, which
can increase the binding affinity between the boric acid group
and glucose at physiological condition.[15] Moreover, pyridinium
salts have been confirmed to have strong binding affinity with
water-soluble pillar[5]arene (WP5) to form stable inclusion
complexes.[16] Accordingly, a pyridylboronic acid derivative (G)
containing a long alkyl chain as the hydrophobic tail was
synthesized for further assembling to form supra-amphiphile with
WP5. Based on the above design, herein, we report a novel
glucose-responsive supramolecular drug delivery system based
on the host−guest interaction between WP5 and a pyridylboronic
acid derivative G, which can successfully encapsulate large
molecular weight insulin with high loading efficiency (Scheme 1).
To the best of our knowledge, this is the first example of small
size supramolecular vesicles with excellent encapsulation
capacity of large protein molecule such as insulin. Moreover, it
was demonstrated that high glucose concentration could
facilitate the quick release of insulin from insulin-loaded
supramolecular nanocarriers, suggesting a smart DDS for the
potential application in diabetic therapy.
water solubility and inclined to self-aggregate in water, a model
guest MG was synthesized (Scheme S2, Supporting
Information) to investigate the host−guest complexation between
WP5 and the guest molecule G via 1H NMR spectroscopy in D2O.
Figure 1 showed the 1H NMR spectra of MG recorded in the
absence and presence of WP5 (1.0 equiv.), and it was found
that after complexation the signals of phenyl protons (Ha) and
methylene protons (Hc) from WP5 shifted downfield slightly.
Whereas, the signals derived from pyridine protons (H1 and H2)
of MG shifted upfield slightly, and butyl protons (H3, H4, H5, and
H6) of MG shifted upfield remarkably and showed obviously
broaden effects due to the shielding effect of the electron-rich
cavities of pillar[5]arene, which suggested the inclusion of the
alkyl chain and partial pyridine group of MG into the hydrophobic
WP5 cavity and the formation of WP5⊃MG inclusion complex.
Figure 1. 1H NMR spectra (400 MHz, D2O, 298 K): (a) MG (10.0 mM); (b) MG
+ WP5; (c) WP5 (10.0 mM).
The stoichiometry of complexation between WP5 and MG in
water was further investigated by the Job’s plot method using
the UV−Vis spectroscopy, which confirmed the 1:1 binding
stoichiometry between WP5 and MG (Figure S8, Supporting
Information). And the association constant (Ka) for WP5⊃MG
was further determined to be (1.55 ± 0.33) × 105 M−1 by using
fluorescent titration experiments (Figure S9, Supporting
Information). The binding affinity for such host−guest inclusion
was mainly driven by the cooperative electrostatic and
hydrophobic interactions. Therefore, we deduced that WP5 and
pyridylboronic acid derivative G could form a “tadpole-like” 1:1
amphiphilic supramolecular inclusion complex as shown in
Scheme 1.
Scheme 1. Schematic illustration of the formation of supramolecular vesicles
based on WP5⊃G supra-amphiphile and their glucose-responsive insulin
release.
Results and Discussion
Construction of supramolecular binary vesicles in water
based on the WP5⊃G supra-amphiphiles: After achieving the
above amphiphilic WP5⊃G supramolecular complex in water,
the ability of such a supra-amphiphile to form higher-order
aggregates in aqueous phase was further investigated. When a
certain amount of WP5 solution was added to the transparent G
solution, a light opalescence and obvious Tyndall effect could be
Host-guest complexation studies in water: WP5 was
synthesized according to the reported literature,[17] and a well-
designed guest molecules G bearing the pyridylboronic acid
motif was successfully synthesized by using pyridin-4-ylboronic
acid as the starting material (Scheme 2). Since G exhibited poor
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observed (Figure 2b inset), indicating the formation of
microaggregates. The morphology and size of these aggregates
were further investigated by transmission electron microscopy
(TEM) and dynamic laser scattering (DLS) experiments. From
the TEM images (Figure 2a), the obtained aggregates showed a
hollow spherical morphology with an average diameter around
120 nm, which was in good agreement with the DLS result
(average diameter of 132 nm, Figure 2b), indicating the
formation of a vesicular structure. Subsequently, the best molar
ratio between WP5 and G for the formation of supramolecular
aggregates was determined to be 10 : 1 ([G]/[WP5]) based on
the UV−Vis spectra[18] (Figure S10, Supporting Information).
Based on this best molar ratio, the critical aggregation
concentration (CAC) for WP5⊃G assembly was determined to
the obtained WP5⊃G vesicles, we first studied their stimuli-
responsive behavior. It was found that the above WP5⊃G
supramolecular vesicles exhibited the characteristic D-glucose-
responsive behavior caused by the strong bonding affinity
between pyridylboronic acid motif and cis-diol-containing D-
glucose, which was also certificated by the 1H NMR and 11B
NMR spectra of MG and WP5⊃MG complex in presence and
absence of D-glucose (Figure S6 and S7, Supporting
Information). After adding 1.0 equiv. of D-glucose to the MG
solution (pD = 7.4), the protons H1−3 of MG shifted upfield
obviously, indicating the binding of MG with D-glucose. Whereas,
when D-glucose (1.0 equiv.) was added to the mixed solution
containing WP5 and MG, protons H3−6 of MG shifted upfield
slightly, which demonstrated the deeper inclusion of the alkyl
be 1 × 10-4 M (Figure S11, Supporting Information). Furthermore, chain of MG into the cavity of WP5 resulted from the tensile
ζ-potential measurements were carried out to examine the
stability of the obtained supramolecular vesicles with different
WP5/G molar ratios, and finally, the molar ratio of [WP5]/[G] =
1/3 (ζ-potential = −38.8 mV, Figure 2c) was chosen for further
investigating their stimuli-responsive behaviors, since the
obtained vesicles under these conditions will not further
assemble into agglomerates due to the repulsive force-induced
increasing stability. Whereas, after loading with cargos, the
molar ratio of [WP5]/[G] = 1/1.5 was used for constructing the
insulin-loaded vesicles, because much more stable nanocarriers
with high insulin-loading efficiency could be obtained under this
condition.
force of D-glucose to MG. Moreover, 11B NMR experiments also
proved the strong bonding between boric acid ligand and D-
glucose for both MG and WP5⊃MG complex, from which
obvious chemical shift changes could be observed after the
addition of D-glucose (Figure S7, Supporting Information).[19]
Furthermore, upon adding D-glucose to the WP5⊃G vesicular
solution (pH = 7.4), no vesicle could be observed from the TEM
image (Figure S13a, Supporting Information) and accompanying
with the disappearence of the Tyndall effect (Figure S13b,
Supporting Information). Moreover, after adjusting the vesicular
solution to pH 6.0, Tyndall effect also disappeared and no
vesiclular structure could be found from the TEM image (Figure
S13c and S13d, Supporting Information) because the generated
pillar[5]arene in its acid form is water-insoluble, which will be
precipitated from the acidic aqueous solution. [3a, 13a, 19b]
Figure 2. (a) TEM images of WP5⊃G aggregates; (b) DLS result of WP5⊃G
aggregates. Inset: Image of the Tyndall effect of WP5⊃G aggregates; (c) the
ζ-potential of WP5⊃G aggregates. ([WP5] = 0.062 mM, [G] = 0.186 mM,
[WP5]/[G] = 1/3).
Figure 3. (a) TEM images of DOX-loaded vesicles formed by WP5 and G; (b)
DLS result of DOX-loaded vesicles. Inset: Images of the Tyndall effect of
DOX-loaded vesicles; (c) Florescence spectra of DOX-loaded vesicles (black
line), DOX-loaded vesicles after the addition of D-glucose (5.5 mg/mL) (red
line), and DOX-loaded vesicles after the solution pH was adjusted to 6.0 (blue
line). ([WP5] = 0.062 mM, [G] = 0.186 mM, [WP5]/[G] = 1/3, pH = 7.4).
Stimuli-responsiveness of the supramolecular vesicles:
Stimuli-responsive properties are often incorporated into
supramolecular nanocarrier systems, which is of great
importance for achieving their controllable drug release.
Therefore, before investigating the insulin loading efficiency of
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Meantime, a model hydrophobic fluorescent dye, doxorubicin
(DOX), was used to further confirm the stimuli-responsive
behaviour of the obtained WP5⊃G vesicles. After being desalted
by TEA, DOX could be successfully loaded into the hydrophobic
Stern layer of the vesicles as confirmed by the TEM images
(Figure 3a) and DLS results (Figure 3b). Moreover, the
fluorescence spectra of DOX-loaded vesicular solution in the
presence and absence of D-glucose were recorded (Figure 3c),
from which the characteristic emission peaks of DOX became
much stronger after the addition of D-glucose, especially with
the extended response time (Figure S14b, Supporting
Information). The same phenomena could also be observed
after adjusting the vesicular solution to pH 6.0 (Figure S14a,
Supporting Information), indicating the collapse of vesicles after
the external stimuli of D-glucose or acidic environment. All the
above results confirmed that such WP5⊃G supramolecular
vesicles exhibited excellent D-glucose- and pH-responsiveness,
which endowed them with the ability to encapsulate insulin at
neutral condition and release them in response to the
environment with high D-glucose concentration or low pH value,
making the obtained WP5⊃G supramolecular vesicles an ideal
candidate for controlled insulin release.
(Figure 4a inset), confirming that insulin molecules were
successfully loaded into the cavities of vesicles. More intuitively,
the vesicular solution became remarkably cloudy and its turbidity
increased significantly after insulin was successfully loaded
(Figure 4b inset).
Meanwhile, the HPLC results of the vesicular solution after
adding Triton X-100 which was used to destroy the
nanoparticles, also revealed that insulin was successfully
encapsulated into the cavity of WP5⊃G vesicles (Figure 5).
Furthermore, the encapsulation efficiency of insulin was
calculated to be 64% based on the peak area detected by HPLC
at ~11 min, which is assigned to the pure insulin peak.
Meanwhile, ζ-potential measurements showed that such insulin-
loaded supramolecular vesicles also had a negative ζ-potential
(−25.6 mV, Figure 4c) with good stability, ensuring their potential
application for insulin delivery and controlled release.
Insulin encapsulation and in vitro release: In the following
study, the insulin encapsulation efficiency of the above resulting
supramolecular vesicles and their release behavior of the loaded
insulin were evaluated. To prepare insulin-loaded vesicles,
aqueous solution of human recombinant insulin was added
quickly to an aqueous solution containing WP5 and G ([G]/[WP5]
= 1.5 : 1, 1% EtOH was added to improve the solubility of G).
After standing overnight, the unloaded insulin was removed by
dialysis against deionized water. TEM images (Figure 4a) and
DLS results (Figure 4b) showed that insulin-loaded vesicles
were much larger in size (~320 nm) than those of the unloaded
vesicles (~132 nm), and the morphology of insulin-loaded
vesicles turned to be irregular shape from the TEM observations
Figure 5. HPLC traces of insulin at 214 nm: (a) retention time of pure insulin;
(b) retention time of insulin released from insulin-loaded WP5⊃G vesicles after
treated with Triton X-100. ([WP5] = 0.118 mM, [G] = 0.177 mM, [WP5]/[G] =
1/1.5).
Due to the intrinsic properties of human recombinant insulin
(without any UV or fluorescence signal), it was difficult to monitor
the release behavior of insulin. Therefore, fluorescein
isothiocyanate (FITC) labeled insulin was used as a model
fluorescent probe as well as an analogous drug to evaluate the
release behavior of insulin in real time. Initially, FITC-insulin-
loaded vesicles were successfully prepared by using the similar
drug-loading process. After adding D-glucose or Triton X-100 to
the obtained drug loaded vesicular solution, the changes in
fluorescence spectra (Figure 6a) indicated that FITC-labeled
insulin was successfully encapsulated into the WP5⊃G vesicles.
Moreover, DLS results (Figure 6b) also confirmed that FITC-
Figure 6. (a) Florescence spectra of FITC-insulin-loaded vesicles in solution
(pH = 7.4, black line), FITC-insulin-loaded vesicles after the addition of D-
glucose (2.0 mg/mL, red line), and FITC-insulin-loaded vesicles after adding
Triton X-100 (blue line). ([WP5] = 0.062 mM, [G] = 0.186 mM, [WP5]/[G] = 1/3,
pH = 7.4); (b) DLS result of FITC-insulin-loaded vesicles.
Figure 4. (a) TEM images of insulin-loaded vesicles formed by WP5⊃G supra-
amphiphile; (b) DLS result of insulin-loaded vesicles formed by WP5⊃G supra-
amphiphile. Inset: Images of WP5⊃G vesicles (right: insulin-loaded vesicular
solution, left: unloaded vesicular solution); (c) the ζ-potential of insulin-loaded
WP5⊃G vesicles. ([WP5] = 0.118 mM, [G] = 0.177 mM, [WP5]/[G] = 1/1.5).
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insulin molecules were successfully loaded into the WP5⊃G
vesicles, which could be concluded from the size changing after
the loading of FITC-insulin. Meanwhile, the size of FITC-insulin-
loaded vesicles was similar with that of human recombinant
insulin loaded vesicles. All the above results indicated that FITC-
insulin could be used as a fluorescent probe as well as an
analogous drug to directly evaluate the release behavior of
insulin in real time.
the MTT cell survival assay against MRC-5 normal cells. Figure
8 shows the cell viability after 24 h of incubation with WP5, blank
vesicles, and FITC-insulin-loaded vesicles at a concentration
varying from 5 to 40 μM. Clearly, the cell viability remains above
70% after 24 h incubation even when the concentration of the
examined vesicles was increased to 40 μM. Therefore, these
supramolecular vesicles that have good cytocompatibility are a
suitable nanocarrier for insulin delivery, which is also essential
for their further biomedical applications.
Subsequently, the release efficiency of FITC-insulin from the
loaded vesicles upon D-glucose stimulus was investigated. As
shown in Figure 7a, almost no FITC-insulin release could be
observed under physiological condition (pH = 7.4). And the
cumulative release amount of insulin was only about 20% within
60 min when the concentration of D-glucose was in normal
blood-glucose level (1.0 mg/mL), indicating that unwanted
insulin release would not happen under the normal blood-
glucose level. However, the encapsulated FITC-insulin was
released rapidly in significant amounts and the cumulative
release amount of FITC-insulin was increased with the
increasing concentration of D-glucose. And the cumulative
release amount of FITC-labeled insulin was about 70% within 60
min when the concentration of D-glucose was increased to 5.5
mg/mL (Figure S15, Supporting Information), indicating
approximately 70% of the loaded FITC-insulin was released
from the cavities of WP5⊃G vesicles. On the other hand, the pH
value also played a vital role in the cargo release behavior. The
FITC-insulin release profiles under neutral (pH = 7.0) and
physiological (pH = 7.4) conditions were presented in Figure 7b,
from which much less FITC-insulin release could be observed
under neutral condition ([D-glucose] = 2.0 mg/mL), whereas the
cumulative release of FITC-insulin reached 60% within 60 min
when the pH value was increased to 7.4 ([D-glucose] = 2.0
mg/mL). Since the normal condition of human blood was weakly
alkaline, the rapid FITC-insulin release could be efficiently
achieved under physiological condition when the blood glucose
concentration became higher, enabling these insulin-loaded
vesicles an ideal candidate for diabetes therapy.
Figure 8. In vitro cytotoxicities of WP5, blank vesicles, and FITC-insulin-
loaded vesicles against MRC-5 cells after 24 h incubation.
Conclusions
In conclusion, we have successfully constructed a novel
glucose-responsive supramolecular drug delivery system based
on the host−guest amphiphilic inclusion complex formed
between
a
water-soluble pillar[5]arene (WP5) and
a
pyridylboronic acid derivative (G) for insulin delivery and
controlled release at physiological condition. The insulin loading
and in vitro drug release experiments demonstrated that insulin
could be successfully encapsulated into the vesicles with high
loading efficiency (64%), which is the first example of small size
supramolecular vesicles with excellent encapsulation capacity of
large protein molecule. Moreover, FITC-labeled insulin was used
as a fluorescent probe as well as an analogous drug to directly
evaluate the release behavior of insulin in real time, which
demonstrated that the insulin-loaded vesicles exhibit excellent
glucose-responsiveness and quick release of insulin at high
glucose concentration environment under physiological condition,
enabling them to be suitable for the potential application in
controlled insulin release. Further cytotoxicity experiments
indicated that these supramolecular vesicles exhibited good
cytocompatibility and they are a suitable nanocarrier for insulin
delivery. The present study opens a novel avenue for the
construction of glucose-responsive smart supramolecular
nanocarriers, which have great potential applications for the
treatment of diabetes mellitus. The in vivo activity of the insulin-
loaded supramolcecular vesicles in treatment of diabetes in a
mouse model is in progress in our laboratory.
Figure 7. The time-dependent FITC-insulin release efficiency of FITC-insulin-
loaded vesicles under different conditions concentrations: (a) in aqueous
solutions (pH = 7.4) containing different concentrations of D-glucose; (b) in
aqueous solutions with the presence of D-glucose (2.0 mg/mL) under different
pH values.
Cytocompatibility of the drug nanocarriers: As we known
that the cytocompatibility of the nanocarrier is also an important
index for evaluating its biomedical application. So the
cytotoxicity of these drug nanocarriers was further evaluated by
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solvent of water (10 mL) and acetone (10 mL) for 48 h in the atmosphere
of Ar. After removing the acetone under vacuum, the water solution was
washed by n-butanol (2 × 20 mL). And the obtained aqueous solution
was concentrated under vacuum to give the target compound MG as a
light yellow solid (78.0 mg, 0.41 mmol, 41%). M. P. 73 – 75 oC. 1H NMR
(400 MHz, DMSO-d6, 298 K) δ (ppm) = 9.12 (s, 2H, OH), 9.04 (d, J = 6.4
Hz, 2H, PyH), 8.31 (d, J = 6.4 Hz, 2H, PyH), 4.59 (t, J = 7.3 Hz, 2H,
PyCH2), 1.94 – 1.86 (m, 2H, PyCH2CH2), 1.30 – 1.24 (m, 2H, CH2CH3),
0.92 (t, J = 7.4 Hz, 3H, CH2CH3). 13C NMR (100 MHz, DMSO-d6, 298 K)
δ (ppm) =143.2, 132.0, 95.5, 60.3, 32.6, 18.8, 13.3. HR-ESI-MS: m/z
Calcd for C9H15NBO2 [M - Br]+ 136.1121, found 136.1120.
Experimental Section
Materials: Human recombinant insulin (Min: ≥27.5 units/mg) was
provided by CCIS Reagent Co., Ltd. (Shanghai, China). FITC-insulin was
provided by Sigma-Aldrich (Shanghai, China). Doxorubicin (DOX•HCl)
(>98%) was provided by Melone Pharmaceutical Co., Ltd. (Dalian, China).
WP5, G, and MG were synthesized and purified according to previously
reported procedures, and they were identified by 1H
&
13C NMR
spectroscopy and mass spectrometry. Cell culture: MRC-5 normal
human cells were cultivated in Dulbecco’s modified Eagle’s medium
(DMEM) supplied with 10% fetal bovine serum (FBS) and antibiotics (50
U·mL−1 penicillin and 50 U·mL−1 streptomycin) at 37 oC in a humidified
atmosphere containing 5% CO2.
DOX loading and release of WP5⊃G vesicles. DOX-loaded vesicles
were prepared as follows: a certain amount of DOX (after being desalted
by TEA) was added to a solution containing WP5 and G (1% EtOH was
added to improve the solubility of G). The ultimate concentrations of DOX,
G, and WP5 were 0.036 mM, 0.186 mM, and 0.062 mM, respectively.
After standing overnight, the prepared DOX-loaded vesicles were purified
by dialysis (molecular weight cutoff 10 000) in distilled water for several
times until the water outside the dialysis tube exhibited negligible DOX
fluorescence.
Synthetic procedures: All reactions were performed in atmosphere
unless noted and all yields were given as isolated yields. WP5
synthesized according to the literature. The detailed synthetic procedures
of pyridine-4-boronic acid derivatives (G and MG) were shown in Scheme
[20]
was
2.
The DOX encapsulation efficiency was calculated by the following
equation.
Encapsulation efficiency (%) = (mDOX-loaded/mDOX) × 100
Where mDOX-loaded and mDOX are mass of DOX encapsulated into the
vesicles and mass of DOX added, respectively. The mass of DOX was
measured by
calculated as relative to
a
fluorescence spectrophotometer at 560 nm and
standard calibration curve in the
a
concentrations from 0.5 to 7.0 μg/mL.
In a typical pH-induced release experiment, a trace amount of 0.01 M
HCl solution was added into 10 mL of DOX-loaded vesicular solution, the
final pH of the vesicular solution was adjusted to 6.0. At selected time
intervals, 4 mL of the release media was taken out for measuring the
released DOX concentrations by the fluorescence technique, and then
was returned to the original release media. The concentration of DOX
was determined by measurement of emission intensity at 560 nm using a
standard emission vs concentration curve constructed for DOX in the
corresponding release medium. A nearly 100% release of DOX from
DOX-loaded vesicles could be obtained by adding Triton X-100 to the
vesicular solution.
In a typical D-glucose-induced release experiment, a certain amount of
D-glucose was added into 10 mL of DOX-loaded vesicular solution, the
final concentration of D-glucose in the vesicular solution was 5.5 mg/mL,
and then the solution pH was adjusted to 7.4. At selected time intervals,
4 mL of the release media was taken out for measuring the released
DOX concentrations by the fluorescence technique, and then was
returned to the original release media. The concentration of DOX was
determined by measurement of emission intensity at 560 nm using a
standard emission vs concentration curve constructed for DOX in the
corresponding release medium. A nearly 100% release of DOX from
DOX-loaded vesicles could be obtained by adding Triton X-100 to the
vesicular solution.
Scheme 2. Synthesis of the pyridine-4-boronic acid derivatives G and MG.
1) Synthesis of compound 1: [21] Pyridine-4-boronic acid (1.2 g, 10.0
mmol) and neopentyl glycol (1.1 g, 10.0 mmol) were dissolved in Toluene
(120 mL). The above mixture was stirred at 130 oC for 12 h, and then the
precipitation was collected by filtration and dried in vacuum. The target
compound 1 was obtained as a white solid (1.6 g, 8.38 mmol, 84%). 1H
NMR (400 MHz, CDCl3, 298 K) δ (ppm) = 8.61 (d, J = 5.6 Hz, 2H, PyH),
7.66 (d, J = 5.6 Hz, 2H, PyH), 3.78 (s, 4H, OCH2), 1.03 (s, 6H, CH3).
2) Synthesis of compound G: Compound 1 (400.0 mg, 2.1 mmol) and
n-hexadecyl bromide (900.0 mg, 3.2 mmol) were dissolved in acetonitrile
(25 mL) and the mixture was refluxed at 80 oC for 72 h. Then the reaction
mixture was cooled down to room temperature, and extracted with
petroleum ether (5 × 15 mL) to remove the unreacted n-hexadecyl
bromide, after removing acetonitrile under vacuum, the residue was
hydrolyzed in a mixed solvent of water (10 mL) and acetone (10 mL) at
60 oC for 24 h. The precipitation was collected, and washed by water,
which was then dissolved in methanol (1 mL) and the obtained solution
was added dropwise to plenty of isopropyl ether (100 mL), the
precipitates were collected by filtration, washed by diethyl ether and dried
in vacuum. The target compound G was obtained as a white solid (500.0
mg, 1.17 mmol, 56%). M. P. 196 – 198 oC. 1H NMR (400 MHz, CD3OD,
298 K) δ (ppm) = 8.83 (d, J = 5.9 Hz, 2H, PyH), 8.20 (d, J = 5.9 Hz, 2H,
PyH), 4.58 (t, J = 7.5 Hz, 2H, PyCH2), 2.03 – 1.99 (m, 2H, PyCH2CH2),
1.38 – 1.28 (m, 26H, CH2), 0.90 (t, J = 6.3 Hz, 3H, CH3). 13C NMR (100
MHz, CD3OD, 298 K) δ (ppm) = 143.7, 133.3, 62.7, 33.2, 30.91, 30.89,
30.8, 30.7, 30.6, 30.2, 27.3, 23.9, 14.6. HR-ESI-MS: m/z Calcd for
C21H39NBO2 [M - Br]+ 304.2999, found 304.3000.
Insulin loading and release of WP5⊃G vesicles. Insulin-loaded
vesicles were prepared as follows: a certain amount of insulin was added
to a solution containing WP5 and G (1% EtOH was added to improve the
solubility of G). The ultimate concentrations of insulin, G, and WP5 were
0.0033, 0.177, and 0.118 mM, respectively. After standing overnight, the
prepared insulin-loaded vesicles were purified by dialysis (molecular
weight cutoff 10 000) in distilled water for several times until the water
outside the dialysis tube exhibited negligible HPLC traces.
3) Synthesis of compound MG: Compound 1 (191.0 mg, 1.0 mmol) and
n-butyl bromide (2.7 g, 20.0 mmol) were dissolved in acetonitrile (30 mL),
and stirred at 60 oC for 48 h in the atmosphere of Ar. Then, the solvent
was removed under vacuum and the residue was hydrolyzed in a mixed
The insulin encapsulation efficiency was calculated by the following
equation.
Encapsulation efficiency (%) = (minsulin-loaded/minsulin) × 100
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10.1002/chem.201700345
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Where minsulin-loaded and minsulin are mass of insulin encapsulated into
the vesicles and mass of insulin added, respectively. The mass of insulin
was detected by HPLC at 214 nm and calculated as relative to a
standard calibration curve in the concentrations from 1.0 to 10.6 μg/mL.
In a complete release experiment, insulin-loaded vesicles prepared as
mentioned above were treated with Triton X-100. After standing at 25 oC
for 10 min, 0.5 mL of sample was withdrawn and analyzed by HPLC
using an AKZONOBEL KR100-5C18 reverse phase column (4.6 × 250
mm) with UV detection at 214 nm at 25 oC. An isocratic elution of
acetonitrile-0.2 M sodium sulfate buffer solution (26 : 74, v/v, phosphoric
acid and ethanol amine were used to adjust pH to 2.3) was applied at a
flow rate of 1 mL•min−1. During assays, 20 μL of sample was injected into
the analytic column and the release of insulin was detected by detector at
214 nm. The encapsulated efficiency of insulin was determined based on
the peak area that detected at ~11 min, which is assigned to the pure
insulin peak.
into each well and the cells were incubated for 4 h. After that, the
medium containing MTT was removed and DMSO (150 μL) was added to
each well to dissolve the formazan crystals. Finally, the plates were
shaken for 10 min, and the absorbance of formazan product was
measured at 490 nm by a microplate reader (BioTek ELx808). Untreated
cells in medium were used as the blank control. All experiments were
carried out with five replicates. The cytotoxicity was expressed as the
percentage of the cell viability relative to the blank control.
Acknowledgements
This work was supported by the National Basic Research
Program of China (2014CB846004), National Natural Science
Foundation of China (No. 21572101, 21472089), and Jiangsu
Provincial Natural Science Foundation of China (BK20140595).
Xiao-Yu Hu also thanks the Alexander von Humboldt Foundation
for the research fellowship.
FITC-insulin loading and release of WP5⊃G vesicles. FITC-insulin-
loaded vesicles were prepared as follows: a certain amount of FITC-
insulin was added to a solution containing WP5 and G (1% EtOH was
added to improve the solubility of G). The ultimate concentrations of
FITC-insulin, G, and WP5 were 0.0033, 0.177, and 0.118 mM,
respectively. After standing overnight, the prepared FITC-insulin-loaded
vesicles were purified by dialysis (molecular weight cutoff 100 000) in
distilled water.
Keywords: supramolecular nanocarrier • insulin delivery •
glucose-responsive • pillararene • controlled release
The encapsulation efficiency of FITC-insulin was calculated based on
the above mentioned equations, which was calculated to be 47%.
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FULL PAPER
Lei Gao, Tingting Wang, Keke Jia, Xuan
Wu, Chenhao Yao, Wei Shao, Dongmei
Zhang, Xiao-Yu Hu* and Leyong Wang*
A glucose-responsive supramolecular
drug delivery system constructed by the
host−guest interaction between a water-
soluble pillar[5]arene (WP5) and a
Page No. – Page No.
pyridylboronic acid derivative (G) was
successfully constructed for insulin
delivery and controlled release at
physiological condition, which provides a
novel strategy for the construction of
glucose-responsive smart supramolecular
drug delivery system and represents the
ideal treatment of diabetes mellitus.
Glucose-Responsive Supramolecular
Vesicles Based on Water-Soluble
Pillar[5]arene and Pyridylboronic Acid
Derivative for Controlled Insulin
Delivery
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