Sugar-Breathing Glycopolymersomes for Regulating Glucose Level

Article abstract of DOI:10.1021/jacs.7b03219

Diabetes mellitus is a chronic, life-threatening illness that affects people of every age and ethnicity. It is a long-term pain for those who are affected and must regulate their blood glucose level by frequent subcutaneous injection of insulin every day. Herein, we propose a noninsulin and antidiabetic drug-free strategy for regulating blood glucose level by a nanosized "sugar sponge" which is a lectin-bound glycopolymersome capable of regulating glucose due to the dynamic recognition between the lectin and different carbohydrates. The glycopolymersome is self-assembled from poly(ethylene oxide)-block-poly[(7-(2-methacryloyloxyethoxy)-4-methylcoumarin)-stat-2-(diethylamino)ethyl methacrylate-stat-(α-d-glucopyranosyl)ethyl methacrylate] [PEO-b-P(CMA-stat-DEA-stat-GEMA)]. The lectin bound in the glycopolymersome has different affinity for the glucose in the blood and the glucosyl group in the glycopolymersome. Therefore, this sugar sponge functions as a glucose storage unit by dynamic sugar replacement: The lectin in the sugar sponge will bind and store the glucose from its surrounding solution when the glucose concentration is too high and will release the glucose when the glucose concentration is too low. In vitro, this sugar-breathing behavior is characterized by a remarkable size change of the sugar sponge due to the swelling/shrinkage at high/low glucose levels, which can be used for blood sugar monitoring. In vivo, this sugar sponge showed an excellent antidiabetic effect for type I diabetic mice within 2 days upon one dose, which is much longer than traditional long-acting insulin. Overall, this concept of "controlling sugar levels with sugar" opens new avenues for regulating the blood glucose level without the involvement of insulin or other antidiabetic drugs.

Full text of DOI:10.1021/jacs.7b03219

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Article
Sugar-Breathing Glycopolymersomes for Regulating Glucose Level
Yufen Xiao, Hui Sun, and Jianzhong Du
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†,ǁ
†,ǁ
†,‡,
Yufen Xiao , Hui Sun and Jianzhong Du *
†
Department of Polymeric Materials, School of Materials Science and Engineering, Tongji University, 4800 Caoan
Road, Shanghai 201804, China. Email: jzdu@tongji.edu.cn; Fax: +86-21-69580239; Tel: +86-21-69580239
‡
Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China.
KEYWORDS: glycopolymersome, polymer vesicle, self-assembly, diabetes mellitus, blood glucose level
ABSTRACT: Diabetes mellitus is a chronic, life-threatening illness that affects people of every age and ethnicity. It is a
long-suffering pain for those who are affected and must regulate their blood glucose level by frequent subcutaneous injec-
tion of insulin every day. Herein, we propose a non-insulin and antidiabetic-drug-free strategy for regulating blood glu-
cose level by a nano-sized “sugar sponge” which is a lectin-bound glycopolymersome capable of regulating glucose due to
the dynamic recognition between the lectin and different carbohydrates. The glycopolymersome is self-assembled from
poly(ethylene oxide)-block-poly[(7-(2-methacryloyloxyethoxy)-4-methylcoumarin)-stat-2-(diethylamino)ethyl methacry-
late-stat-(α-D-glucopyranosyl)ethyl methacrylate] [PEO-b-P(CMA-stat-DEA-stat-GEMA)]. The lectin bound in the glyco-
polymersome has different affinity for the glucose in the blood and the glucosyl group in the glycopolymersome. There-
fore, this sugar sponge functions as a glucose storage by dynamic sugar replacement: The lectin in the sugar sponge will
bind and store the glucose from its surrounding solution when the glucose concentration is too high, and will release the
glucose when the glucose concentration is too low. In vitro, this sugar-breathing behavior is characterized by a remarka-
ble size change of sugar sponge due to the swelling/shrinkage at high/low glucose level, which can be used for blood sug-
ar monitoring. In vivo, this sugar sponge showed excellent antidiabetic effect to type I diabetic mice within two days upon
one dose, which is much longer than traditional long-acting insulin. Overall, this sugar sponge opens new avenues for
regulating the blood glucose level without the involvement of insulin or other antidiabetic drugs.
INTRODUCTION
of four essential submits. In solution, Con A consists
largely of dimers below pH 6 while tetramers above pH 7.
Both negatively and positively charged Con A show high
affinity for glucose, mannose and their derivatives while
the Oδ1 and Oδ2 of asparagine accept hydrogen bonds
from 6-OH and 4-OH of sugar, whereas Nδ2 of asparagine
Diabetes mellitus is a chronic disease characterized by
disorder of glucose regulation. It is predicted that by 2030
the total number of diabetes will reach 366 million. Insu-
1
lin is a peptide hormone produced by β cells of the pan-
creatic islets. It regulates the blood glucose level by pro-
moting the absorption of glucose and its conversion into
1
5,16
donates such a bond to 4-OH of sugar.
Also, sugars
2
play fundamental and crucial biological roles in nature.
The cell-specific targeting based on carbohydrate-protein
interaction, i.e., sugars and lectins, is important for cell-
cell and cell-matrix communication, which in turn con-
tributes to cell recognition, microbial pathogenesis and
glycogen. The treatment of type I diabetes mellitus usu-
ally involves frequent subcutaneous injection of insulin
every day, which is inconvenient and accompanied with
pain, local tissue necrosis, infection, nerve damage, and
difficulty in achieving postprandial blood glucose control,
1
5,17,18
3
immunological recognition, etc.
Like nucleic acids
etc. For example, rapid-acting insulin needs to be inject-
and peptides, sugars have recently gained much attention
ed before meals, while even for long-acting insulin, daily
injection before sleep is necessary to imitate the basal
insulin secretion. To solve these problems, non-invasive
methods were developed and glucose-responsive boric-
acid-based polymers and hydrogels were used for con-
trolled release of insulin.
controlling the blood sugar still involve insulin, which
may result in side effects such as hypoglycemia, insulin
resistance, weight gain, allergy and hypokalemia, etc.
1
9,20
because of their significance and abundance.
Further-
more, sugar-containing copolymers are becoming increas-
ingly important due to their promising potential biomed-
21-23
ical applications.
Usually, the weak protein-sugar in-
4-12
teractions can be significantly strengthened by the multi-
valent effect of clustered sugars (cluster glycoside
However, such systems for
2
4,25
effect).
Recently, much effort has been paid to develop
25-28
13,14
sugar-containing polymers for cell sensing,
therapeu-
2
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33-36
tics,
and synthetic biology, etc.
Lectins are proteins capable of specific binding to car-
bohydrates. Concanavalin A (Con A) is a lectin composed
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Polymer vesicles, also referred to as polymersomes, glucose-coated polymersome using dynamic covalent
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have attracted much attention because of their unique
structure and diverse potential applications in biophar-
maceutical field, especially for stimuli-responsive vesi-
bond between sugar and lectins.
Herein, a non-insulin and antidiabetic-drug-free strate-
gy is proposed to regulate the blood glucose level of dia-
betes based on a lectin-bound glycopolymersome. This
sugar sponge can store extra glucose when the concentra-
tion is higher than normal level and release it when the
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,37,38
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cles.
External stimuli, such as pH, temperature,
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light, redox, ultrasound, and enzyme,
have been
applied to trigger the responsiveness of “smart vesicles”
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for various applications in drug delivery,
gene deliv-
concentration is too low, demonstrating
a sugar-
4
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49,50
16,51
ery,
antibacterial,
contrast enhancement,
etc.
breathing behavior (Scheme 1). This sugar sponge is also
functionally similar to an artificial glycogen as it can
store/release glucose at high/low glucose concentrations.
Furthermore, the in vivo test showed excellent antidiabet-
ic effect towards type I diabetic mice within two days up-
Recently, more and more concerns have been drawn to
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glycopolymersomes.
For example, Schlaad et al.
put forward a glycopolymersome by using “click” reaction
between vinyl-containing polymer and thiol-substituted
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glucose. Alexander and co-workers reported a glycopol-
on one dose of the sugar sponge.
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ymersome with “sweet talking” ability with bacteria.
Lecommandoux et al. fabricated a polymersome by em-
ploying polysaccharide-block-polypeptide copolymer to
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mimic synthetic viral capsids. Jiang’s group prepared a
Scheme 1. Preparation of Sugar Sponge and its Blood Glucose Regulation Behavior via Sugar-Breathing
a
a
Con A can be enriched on the glycopolymersome due to the electrostatic interaction with PDEA, bound with the glucosyl to
form a Con A-bound glycopolymersome (sugar sponge), then immobilized in the membrane of the glycopolymersome by the
photo-cross-linking of CMA under UV irradiation to form cross-linked sugar sponge. The sugar sponges could bind/release glu-
cose at high/low glucose concentrations. The sugar sponges are capable of long circulation for regulating blood glucose level.
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Scheme 2. Synthesis of Monomers and PEO-b-P(CMA-stat-DEA-stat-GEMA) Copolymer
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Glycopolymersome
To prepare sugar sponges, a glycopolymer, [PEO-b-
P(CMA-stat-DEA-stat-GEMA)], was synthesized via atom
transfer radical polymerization (ATRP) and self-
assembled into glycopolymersome. The hydrophobic
CMA and DEA moieties form the membrane, whereas the
biocompatible and hydrophilic PEO chains form the co-
ronas. The GEMA moieties with a glucosyl residue are
embed within the membrane because of the constraints
of the hydrophobic polymer chain. Con A will be bound
in the membrane of the glycopolymersome when it is
added into the polymersome solution due to the electro-
static effect with DEA, thus assists the recognition inter-
actions between Con A and glucosyl, leading to the for-
mation of the un-cross-linked sugar sponge. The photo-
cross-linking of CMA under UV irradiation in the mem-
brane of the glycopolymersome can stabilize its structure
and prevent its disassociation upon dilution or environ-
mental change. Also, this process can immobilize Con A
in the membrane of the glycopolymersome to afford the
cross-linked sugar sponges with longer circulation time in
vivo.
RESULTS AND DISCUSSION
Synthesis of Glycopolymer PEO-b-P(CMA-stat-
DEA-stat-GEMA). The glycopolymer PEO-b-P(CMA-
stat-DEA-stat-GEMA) was synthesized by ATRP, followed
by the deprotection of the precursor PEO-b-P(CMA-stat-
DEA-stat-AcGEMA) copolymer in the presence of sodium
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methylate. The synthetic details and the corresponding
1
H NMR spectra of PEO-Br, CMA and AcGEMA mono-
mers, glycopolymer precursor PEO-b-P(CMA-stat-DEA-
stat-AcGEMA) and glycopolymer are provided in Figures
S1-S6 in the Supporting Information. The degrees of
polymerization (DPs) of CMA, DEA and AcGEMA are 8,
8
0 and 12, as calculated from Figure S5 and Table S1 in the
Supporting Information. As shown in Figure S6A, the dis-
appearance of peaks in the high field revealed the suc-
cessful removal of acetyl group (comparing with Figure S5
in the Supporting Information), while the intensity of
other peaks remained the same, suggesting successful
synthesis of the glycopolymer. The gel permeation chro-
matography (GPC) trace of this glycopolymer is shown in
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Figure S7, showing a narrow molecular weight distribu- the polydispersity (PD) is 0.091 (Figure S8 in the Support-
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tion (M = 9700 Da and M /M = 1.17).
ing Information), considering the collapse at dry state and
the hydration in aqueous solution. The membrane thick-
ness is ca. 24 nm (Figure 1F), which is consistent with the
theoretical value (25.1 nm) as calculated in Scheme S1 in
the Supporting Information.
n
w
n
Self-assembly
of PEO-b-P(CMA-stat-DEA-stat-
GEMA) into Glycopolymersomes. The glycopoly-
mersome was prepared by self-assembly of PEO -b-
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P(CMA -stat-DEA -stat-GEMA ) glycopolymer in the
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mixture of THF/H O (1:3, v/v) at an initial concentration
The ζ-potential of glycopolymersome is + 46.6 mV
(Figure S9 in the Supporting Information), indicating that
the hydration of GEMA in the glycopolymersome mem-
brane facilitates the solubility of the DEA block in water.
The positively charged glycopolymersome can interact
with negatively charged Con A and then facilitate the
recognition and binding between Con A and GEMA. Also,
both hydrophilic PEO coronas and strong positive charges
can maintain the excellent stability of glycopolymersomes
in water, which is important for their long circulation in
vivo.
2
(
C ) of 2.0 mg/mL in THF. THF was then removed by
ini
1
dialysis. H NMR study of the glycopolymersome in D O
2
(
Figure S6B) reveals the structure of glycopolymersome.
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The signal from the hydrophilic PEO chains suggests that
they form the coronas of the glycopolymersome. The at-
tenuated signal from the whole P(CMA -stat-DEA -stat-
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GEMA ) block indicates that it forms the membrane of
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the glycopolymersome along with a fraction of hydro-
philic PGEMA moieties embedded in the membrane. Full
discussions on the NMR analysis of the glycopoly-
mersomes are provided in the Supporting Information
Immobilization of the Vesicular Structure of Gly-
copolymersomes by Photo-Cross-Linking of Couma-
rin Moieties. To immobilize the vesicular structure of
glycopolymersomes, the coumarin group was introduced
to the glycopolymer, which can undergo photo-
dimerization upon UV irradiation (λ ≈ 365 nm) to afford
inter-chain covalent bonds (Figure 2). TEM images in
Figure 3 revealed that the photo-cross-linking procedure
didn’t change the structure of the glycopolymersomes.
DLS studies indicated that the photo-cross-linking proce-
dure didn’t change the size of glycopolymersomes (Figure
S8). Besides, the UV-vis spectroscopy was employed to
monitor the photo-cross-linking procedure. Figure 2
showed that the degree of cross-linking increased with
the exposure time under UV irradiation, suggesting that
the degree of cross-linking can be precisely controlled by
adjusting the exposure time.
(
Figure S6). The structure of the glycopolymersome is
schematically presented in Scheme 2.
Figure 1. Electron microscope studies of un-cross-linked gly-
copolymersomes: (A) and (B) TEM images of glycopoly-
mersomes stained by neutral phosphotungstic acid; (C) TEM
images of Con A-bound glycopolymersome (un-cross-linked
sugar sponge); (D) and (E) SEM images of un-cross-linked
glycopolymersomes; (F) Simulation of a totally collapsed
glycopolymersome in the SEM study, suggesting a membrane
thickness of 24 nm. The short red lines in E and F indicate
two layers of membrane (2d).
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.2
.0
.8
.6
.4
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5 s
30 s
60 s
120 s
240 s
Furthermore, TEM and SEM were applied to reveal the
morphology of glycopolymersomes. As shown in Figure 1A
and B, the TEM images confirmed a classical collapsed
membrane structure of glycopolymersomes. As men-
tioned before, the hydration of the embedded glucose
moieties decreased the density of the membrane, result-
ing in the softening of the glycopolymersomes. Therefore,
the glycopolymersomes were liable to collapse from dif-
ferent directions. This phenomenon was further verified
by the SEM studies in Figure 1 where some bowl-shaped
dry glycopolymersomes with a fully collapsed membrane
structure (from top to bottom) and partially collapsed
glycopolymersomes were visible. The average diameter of
the glycopolymersomes is ca. 390 nm (Figure 1A), which is
consistent with the dynamic light scattering (DLS) analy-
480 s
960 s
280
300
320
340
360
Wavelength (nm)
Figure 2. UV-vis spectra of photo-cross-linked glycopoly-
mersomes with different degrees of cross-linking.
sis where the hydrodynamic diameter (D ) is 362 nm and
h
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bigger due to the binding of Con A in the membrane of
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the glycopolymersomes.
It is noteworthy that the Con A binds the glucosyl
groups only within the individual glycopolymersomes
instead of inter-glycopolymersomes. Otherwise, the di-
ameter of the glycopolymersomes would be expected to
greatly increase. Therefore, there is little chance for Con
A to bridge several glycopolymersomes together since the
glucosyls were embedded into the membrane. Further-
more, Sepharose column was used to purify the un-cross-
linked sugar sponge to remove free Con A (read details of
that in the Supporting Information). After flowing
through Sepharose column, the size of the un-cross-
linked sugar sponge almost unchanged (Figure S11 in the
Supporting Information), further confirming that the Con
A was immobilized within the glycopolymersome.
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Figure 3. TEM images of cross-linked glycopolymersomes
without Con A binding.
Preparation of Sugar Sponges and Study on the
Carbohydrate-Lectin Interactions. The stability of the
un-cross-linked glycopolymersomes in HEPES buffer was
evaluated first as this is the solution for sugar sponge
preparation. As shown in Figure 4A, the diameter of the
un-cross-linked glycopolymersomes barely changed at
different concentrations of HEPES buffers, indicating the
ionic strength did not influence the stability of glycopol-
ymersomes. Then, the Con A-bound glycopolymersomes
In order to determine the loading efficiency of FITC-
Con A on the glycopolymersomes, the calibration curve of
FITC-Con A in HEPES buffer was plotted by fluorescence
spectrometry (Figure S12). The fluorescence intensity of
the un-cross-linked sugar sponge was 5797 at 520 nm af-
ter Sepharose column (Figure S13), corresponding to a
Con A loading efficiency of 34%. The electrostatic interac-
tions between DEA moieties and FITC-Con A contributed
the high loading efficiency, which can be confirmed by
Zeta potential studies before and after Con A binding (the
ζ value decreased from + 46.6 mV to + 10.8 mV after Con
A binding, see Figure S9).
(
un-cross-linked sugar sponge) were prepared by the
recognition between Con A and glucosyl of glycopolymer
based on the carbohydrate-lectin interaction. The un-
cross-linked sugar sponge with a concentration of FITC-
Con A of 75 μg/mL was exposed under UV irradiation for
(A)
650 (B)
600
D (nm) PD
(a) 0 mM
(b) 5 mM
(c) 10 mM
(d) 15 mM
(e) 20 mM
h
(
(
(
(
a) 362 0.091
b) 340 0.194
c) 362 0.196
d) 366 0.216
Con A
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seconds to give a degree of cross-linking of 50% (Figure
). The inter-chain covalent bonds ensured the stability of
550
500
the vesicular structure and immobilized Con A on the
membrane but did not entirely fix the structure of the
(e) 352 0.204
450
400
350
glycopolymersome
to
provide
excellent
swell-
100
1000
0
50
100
150
200
g/mL
ing/shrinkage properties. Thus the photo-cross-linked
Con A-bound glycopolymersome could be regarded as a
cross-linked sugar sponge.
Hydrodynamic Diameter (nm)
Concentration of FITCꢀCon A
(µ
)
4
00
80
1.0 400
0.8 380
0.6 360
0.4 340
0.2 320
0.0 300
1.0
0.8
0.6
0.4
0.2
0.0
(C) unꢀcrossꢀlinked glycopolymersome
(D) crossꢀlinked glycopolymersome
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360
Lectins are proteins capable of specific binding to car-
bohydrates. Con A is a lectin with high affinity for glucose
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20
1
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and mannose. Herein, the un-cross-linked FITC-Con A-
bound glycopolymersome (un-cross-linked sugar sponge;
here FITC is fluorescein isothiocyanate) was utilized to
demonstrate the specificity of carbohydrate-lectin inter-
actions. DLS was applied to monitor the recognition be-
tween FITC-Con A and glycopolymersomes. Figure 4B
revealed that the size of glycopolymersomes increased
with the concentration of FITC-Con A due to the for-
mation of Con A-bound glycopolymersome by the recog-
nition of the glucosyl embedded in the membrane and
the FITC-Con A in the solution. For example, the diame-
ters of this sugar sponge are 375, 390, 425, 465, 504, 522
and 537 nm when the concentration of FITC-Con A in-
creases from 50 to 200 μg/mL (Figure 4B and Figure S10A).
TEM was applied to reveal the morphology of the un-
cross-linked sugar sponge (Figure 1C). Comparing with
the un-cross-linked glycopolymersomes (without Con A)
in Figure 1A, the membrane thickness of the un-cross-
linked sugar sponge (glycopolymersomes with Con A) is
300
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Concentration of Glucose (mg/mL)
Concentration of Glucose (mg/mL)
Figure 4. DLS studies of glycopolymersomes before and
after cross-linking: (A) Stability of un-cross-linked glyco-
polymersomes against HEPES buffers; (B) Carbohydrate-
lectin interactions between un-cross-linked glycopoly-
mersomes and Con A as revealed by the variation of hy-
drodynamic diameters; (C) and (D) confirm that no glu-
cose-breathing behavior for un-cross-linked (C) and cross-
linked (D) glycopolymersomes without Con A binding.
Sugar Breathing Study of Sugar Sponges Based on
Carbohydrate-Lectin Interactions. In order to verify
that the sugar breathing behavior is only related to the
carbohydrate-lectin interactions, control experiments
were performed first using the un-cross-linked and cross-
linked glycopolymersomes without the binding of Con A.
As shown in Figure 4C and D, the diameters of the glyco-
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polymersomes stay almost constant at different concen- cose because of the carbohydrate-lectin interactions
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trations of glucose, which means both the un-cross-linked
and cross-linked glycopolymersomes (without Con A em-
bedded in the membrane) in glucose do not have the sug-
ar-breathing function. By contrast, both the un-cross-
linked and cross-linked glycopolymersomes with FITC-
Con A embedded in the membrane possess a similar
(Figure 5A and C). Thus, both the un-cross-linked and
cross-linked Con A-bound glycopolymersomes (un-cross-
linked and cross-linked sugar sponges) have sugar breath-
ing functions, but the cross-linked sugar sponge is sup-
posed to have a longer circulation time due to its better
stability.
“
breathing” behavior at different concentrations of glu-
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Figure 5. DLS and TEM studies of sugar sponges without and with cross-linking: Glucose breathing behavior of the un-
cross-linked sugar sponge (A) and the cross-linked sugar sponge (C) as revealed by the variation of hydrodynamic diame-
ters; (B) Reversibility study of cross-linked sugar sponges determined by the variations in the hydrodynamic diameters
when the glucose concentrations were switched between 0.5 mg/mL and 1.25 mg/mL; (D) Corresponding schematic il-
lustration of breathing behavior; (E-F) TEM images of one cross-linked sugar sponge (Ccon A = 75 μg/mL) in the absence
(
E) and in the presence (F) of glucose (Cglucose = 0.75 mg/mL), showing a remarkable sugar-induced swelling behavior of
sugar sponge.
The Con A binds the glucosyl in the sugar sponge.
cross-linked sugar sponges have mannose-breathing func-
tions, as confirmed by the swelling of sugar sponges with
mannose. However, those sugar sponges can be still used
for monitoring blood sugar levels as only small fraction of
mannose exists in vivo (the ratio of mannose to glucose is
ca. 1:100) and mannose undergoes fast clearance from
However, in the presence of glucose, the Con A will bind
the glucose instead of the glucosyl due to the Con A’s
higher affinity for glucose than for glucosyl. As shown in
Figure 5C and Figure S10B in the Supporting Information,
the size of the cross-linked sugar sponge increases from
5
9
3
90 to 701 nm as the concentration of glucose increases
blood (t1/2 = 30 min). Furthermore, extra mannose in the
blood can be stored in the sugar sponge and released
when needed since mannose cannot be metabolized di-
rectly.
from 0 to 1.5 mg/mL. TEM images of the cross-linked sug-
ar sponge before (Figure 5E and Figure S14) and after glu-
cose binding (Figure 5F and Figure S15) also confirmed
the swelling of the cross-linked sugar sponge after bind-
ing glucose (See Figures S14-S15 in the Supporting Infor-
mation for more TEM images).
The blood glucose level in human body is tightly regu-
lated at a fluctuant range of 3.9 ~ 6.1 mM (0.7 ~ 1.1
mg/mL). TEM images show that the diameter of the
cross-linked sugar sponge is around 600 nm when the
concentration of glucose is 0.75 mg/mL (Figure 5F and
Figure S15). These TEM images also confirm decreased
membrane density of the cross-linked sugar sponge, indi-
cating its tunable permeability. As the concentration of
the glucose increases to a level higher than the normal
blood glucose level (1.25 mg/mL), the cross-linked sugar
sponge will absorb extra glucose. Figure 5D illustrated
Considering the significant degree of swelling of sugar
sponges with glucose (Figure 5 A and C), those sugar
sponges can be applied in glucose detection. Since man-
nose can be produced from glucose in vivo and this may
be a confounding issue in glucose detection, it was neces-
sary to evaluate the breathing behavior of the sugar
2,58
sponges against mannose.
the Supporting Information, both un-cross-linked and
As shown in Figure S16 in
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serious damage to related organs. Therefore, type I diabe-
this binding process: the glucose will diffuse into the
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membrane and bind Con A, substituting the glucosyl on
the polymer chain because of competitive effect, resulting
in swelling of the cross-linked sugar sponge. Consequent-
ly, the diameter of the cross-linked sugar sponge increas-
es to around 650 nm (Figure 5B). On the contrary, the
glucose will be released from the cross-linked sugar
sponge to the solution when the concentration of sugar
decreases. This responsive glucose release was studied in
vitro. As shown in Figure S17 in the Supporting Infor-
mation, the glucose release content is lower compared
with free glucose at the same concentration.
tes model was developed for the in vivo test of the sugar
sponge.
In order to build the type I diabetic mice model, 31 male
KM mice were injected intraperitoneally with streptozo-
cin (120 mg/kg) in citric buffer after fasting for 12 h. A
week later, the weight of the mice changed from 40.8
.5 g to 38.8 3.1 g, while the blood glucose level in-
creased from 6.8 1.4 mmol/L to 22.3 3.5 mmol/L. The
±
2
±
±
±
mice with blood glucose level of 18 – 26 mmol/L were re-
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61
garded as diabetic mice, giving a modeling success rate
of 74%.
40
The in vivo studies of the antidiabetic efficacy of sugar
sponge were performed using these diabetic KM mice.
We monitored the blood glucose level of the diabetic
mice at 2, 6, 12, 24, 36, 48 and 75 h after injection of dif-
ferent solutions. In 2 h, the buffer in the solutions inject-
ed into the caudal vein is almost metabolized, so the dilu-
tion effect of the solution injected barely influences the
Normal mice with HEPES buffer
Diabetic mice with glycopolymersome
Diabetic mice with sugar sponge
Diabetic mice with HEPES buffer
Diabetic mice with Con A
35
30
25
20
62
15
blood glucose level. The second measurement point is 6
h (after fasting 2 h) because the blood glucose level after
10
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63
fasting for 2 h reflects the true influence of eating. Since
the focus of this study is the long-term antidiabetic effect,
we did not monitor the glucose level frequently, instead
we set the measurement interval for 12 h or more.
0
0
10
20
30
40
50
60
70
Time (h)
The diabetic mice were treated with the following solu-
Figure 6. Antidiabetic test of HEPES buffer, glycopoly-
mersome, Con A and sugar sponge to KM mice.
tions: (
and ( ) sugar sponge. The normal mice treated with (5)
HEPES buffer were regarded as the control. In the first 2
hours, the blood glucose levels in groups and 5 slight-
ly descended because of the injection of solutions (Figure
). However, as time went by (for 6 h), the blood glucose
1) HEPES buffer; (2) glycopolymersome; (3) Con A;
4
Cytotoxicity of Glycopolymersome. The cytotoxicity
of glycopolymersome against normal liver L02 cells was
studied (Figure S18 in the Supporting Information) as the
sugar sponges contains toxic coumarin. However, the
glycopolymer only contains 9.8 wt.% of coumarin moie-
ties. The toxic coumarin monomer is polymerized and it
eventually becomes one part of the glycopolymersomes
for long circulation in blood. It is noteworthy that the
hydrophobic coumarin moieties are embedded in the
membrane of glycopolymersomes, preventing the contact
with cells. Furthermore, no coumarins would be released
from the glycopolymersome. Therefore, in principle, the
glycopolymersomes should not be toxic. In reality, the
relevant cell viability against normal liver L02 cells is
around 80 % when the concentration of glycopoly-
mersome is 1000 μg/mL (Figure S18), suggesting low cyto-
toxicity of glycopolymersomes against human cells.
1, 2
6
level of mice rose to the initial level before injection, indi-
cating that the injection of glycopolymersome and HEPES
buffer only slightly affected the blood glucose level in a
very short time. By contrast, the blood glucose level of
mice dramatically decreased in 2 hours in groups 3 and
4.
But for group , the duration of efficacy is very short due
3
to its rapid proteolysis by degradation, as confirmed by
the quick recovery of the blood glucose level after 6 h.
Moreover, the mice died after 36 h due to the high cyto-
toxicity of Con A. Notably, the blood glucose level of dia-
betic mice dropped to normal range after the injection of
sugar sponge, and maintained at about 11 mM at least 36 h,
and increased gradually in 3 days. Comparing to free Con
A, the sugar sponge showed long-term efficacy of regulat-
ing blood glucose level of diabetic mice and greatly re-
duced the cytotoxicity of Con A.
In Vivo Antidiabetics Test. Although both type I and
type II models can be used to evaluate the antidiabetic
effect of the sugar sponge as it can regulate the blood glu-
cose level independent of the type of diabetes mellitus, it
is necessary to compare the antidiabetic effect of our sug-
CONCLUSION
ar sponge with insulin in proper model. Type I diabetes
In conclusion, a novel glycopolymer, PEO-b-P(CMA-stat-
DEA-stat-GEMA), was synthesized and self-assembled
into glycopolymersomes. The PDEA segment can enhance
the carbohydrate-lectin interactions, resulting in a high
loading efficiency of Con A (34%) on the glycopoly-
mersomes. Those Con A-bound glycopolymersomes are
sugar sponges and functionally similar to artificial glyco-
gen. The photo-cross-linking of coumarin moieties fixes
3,60
occurs in about 5-15 percent of all the diabetics.
It is
insulin-dependent and relies on the frequent subcutane-
ous injection of insulin to regulate blood glucose level.
People with type I diabetes are seriously ill from sudden
symptoms of high blood sugar. The high blood sugar
phenotype is stable in this model. Compared with type II
diabetes, type I diabetes lasts lifelong and makes more
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the structure of the glycopolymersome and immobilizes
Page 8 of 10
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the Con A within the membrane of glycopolymersome to
afford the cross-linked sugar sponge. Those sugar sponges
possess excellent reversible glucose responsiveness, show-
ing a glucose regulating behavior: At high glucose con-
centration, the sugar sponges “breathe in” and bind glu-
cose into the membrane, resulting in the swelling of the
sugar sponges. By contrast, the sugar sponges “breathe
out” glucose and shrink at low glucose concentrations.
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can be used for monitoring glucose levels. Moreover, the
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I
diabetic mice within two days upon one dose of sugar
(
sponge. Overall, our study demonstrates a new insight for
regulating blood glucose level without using insulin or
other hypoglycemic drugs, which opens new avenues for
the treatment of diabetes mellitus.
4
(
ASSOCIATED CONTENT
(19) You, L. C.; Schlaad, H. J. Am. Chem. Soc. 2006, 128, 13336-
Supporting Information. The experimental section and
characterization of CMA monomer, GEMA monomer and
glycopolymer; DLS and Zeta potential studies, TEM images,
determination of loading efficiency of FITC-Con A, etc. This
material is available free of charge via the Internet at
http://pubs.acs.org.
13337.
(
20) Sun, P. F.; He, Y.; Lin, M. C.; Zhao, Y.; Ding, Y.; Chen, G.
S.; Jiang, M. ACS Macro Lett. 2014, 3, 96-101.
21) Su, L.; Zhang, W. Y.; Wu, X. L.; Zhang, Y. F.; Chen, X.; Liu,
G. W.; Chen, G. S.; Jiang, M. Small 2015, 11, 4191-4200.
22) Basuki, J. S.; Esser, L.; Duong, H. T.; Zhang, Q.; Wilson,
(
(
P.; Whittaker, M. R.; Haddleton, D. M.; Boyer, C.; Davis, T. P.
AUTHOR INFORMATION
Corresponding Author
Chem. Sci. 2014, 5, 715-726.
(23) Striegler, C.; Schumacher, M.; Effenberg, C.; Müller, M.;
Seckinger, A.; Schnettler, R.; Voit, B.; Hose, D.; Gelinsky, M.;
*
Eꢀmail: jzdu@tongji.edu.cn
Appelhans, D. Macromol. Biosci. 2015, 15, 1283-1295.
(
24) Munoz, E. M.; Correa, J.; Fernandez Megia, E.; Riguera, R.
Author Contributions
J. Am. Chem. Soc. 2009, 131, 17765-17767.
(25) Munoz, E. M.; Correa, J.; Riguera, R.; Fernandez Megia, E.
J. Am. Chem. Soc. 2013, 135, 5966-5969.
ǁ
Y.X. and H.S. contributed equally.
Notes
The authors declare no financial competing interest.
(26) Voskuhl, J.; Stuart, M. C. A.; Ravoo, B. J. Chem. Eur. J.
2010, 16, 2790-2796.
ACKNOWLEDGMENT
(27) Ting, S. R. S.; Min, E. H.; Escale, P.; Save, M.; Billon, L.;
Stenzel, M. H. Macromolecules 2009, 42, 9422-9434.
J.D. is supported by NSFC (21374080, 21674081 and
21611130175), Shanghai International Scientific Collaboration
Fund (15230724500), Shanghai 1000 Talents Plan (SH01068),
and the Fundamental Research Fund for the Central Univer-
sities (1500219107). We appreciate Prof. Yao Sun in The Affili-
ated Stomatology Hospital of Tongji University, Prof. Shish-
eng He, Dongdong Wang and Shaobo Xue in Shanghai Tenth
People’s Hospital for their support in the animal test.
(
28) Park, S.; Kim, G. H.; Park, S. H.; Pai, J.; Rathwell, D.; Park,
J. Y.; Kang, Y. S.; Shin, I. J. Am. Chem. Soc. 2015, 137, 5961-5968.
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Prosperi, D.; Li, W.; Huang, X. F. J. Am. Chem. Soc. 2010, 132,
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3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
ACS Paragon Plus Environment