Supplemental Digital Content 1 - Materials and Methods
Materials
-N-Hydroxysuccinimidyl--maleimidyl poly(ethylene glycol) (NHS-PEGMal, Mw: 5000) was purchased from Nektar Therapeutics (San Carlos, USA). 1, 2distearyl-sn-glycerol-3-phosphatidylethanolamine (DSPE) was purchased from NOF
Corporation (Tokyo, Japan). Sulfo-EMCS was purchased from Pierce Inc (Rockford,
IL). Dichloromethane, chloroform, dimethyl sulfoxide (DMSO), diethyl ether,
triethylamine, streptozotocin (STZ), and tri-sodium citrate dehydrate were purchased
from Nacalai Tesuque (Kyoto, Japan). Fluorescein isothiocyanate (FITC) and Hoechst
33342 were purchased from Dojindo Laboratories (Kumamoto, Japan). Alexa 488labeled goat anti-guinea pig IgG, HEPES buffer solution, and Medium 199 were
purchased from Invitrogen Co. (Carlsbad, USA). Fetal bovine serum (FBS) was
purchased from BioWest (Miami, USA) and phosphate-buffered saline (PBS) was
purchased from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Enzyme-linked
immunosorbent assay (ELISA) kits for the mouse insulin assay were purchased from
Shibayagi Co., Ltd. (Gunma, Japan). Goat normal serum and polyclonal guinea pig
anti-insulin were purchased from Dako (Glostrup, Denmark). 10% formalin solution,
dithiothreitol (DTT), and Triton X-100 were purchased from Wako Pure Chemical
(Osaka, Japan). Ficoll PM400 and NAP-5 column were purchased from GE health
care (GE Healthcare UK Ltd., Buckinghamshire, UK). Collagenase (type S-1) was
purchased from Nitta Gelatin Inc (Osaka, Japan). Conray400 was purchased from
Daiichi Sankyo Healthcare Co., Ltd (Tokyo, Japan). Fibrinogen (type I), plasminogen
from human plasma, PolyA20 and polyT20 carrying (CH2)6-SS-(CH2)6-OH at 5’ end
were purchased from Sigma–Aldrich Chemical Co (St. Louis, Missouri). Thrombin
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was purchased from Kayaku Co., Ltd. (Saitama, Japan). Urokinase was purchased
from American Research Products, Inc (Belmont, MA).
Synthesis of polyDNA-PEG-phospholipid conjugate and polyA-urokinase
Mal-PEG-lipid was synthesized by first dissolving NHS-PEG-Mal (200 mg),
triethylamine (50 μL), and DSPE (20 mg) in dichloromethane and stirring for 48 h at
room temperature (RT) [20,21]. After precipitation with diethyl ether, Mal-PEG-lipid
was obtained as a white powder (190 mg, yield 80%). PolyT20-PEG-lipid (structure
shown in Scheme 1a) was prepared from Mal-PEG-lipid and polyT-SH. PolyT-SH
was obtained by reduction of the disulfide bond of polyT-(CH2)6-SS-(CH2)6-OH with
DTT according to the manufacturer’s instructions. Briefly, the polyT-disulfide
conjugate (in 10mM Tris-HCl, 1mM EDTA pH 8.0) was mixed with DTT (0.04 M)
for 16 h at room temperature (RT) for removal of protection group for thiol. PolyTSH was purified using a NAP-5 column. The SH groups at the 5-ends of polyT were
used to form a conjugate with the Mal-PEG-lipid. A PBS solution of polyDNA-SH
(1.0 mg) was mixed with Mal-PEG-lipid (5.0 mg and the reaction mixture was left for
24 h at RT to form the conjugation, PolyT20-PEG-lipid. PolyT20-PEG-lipid (2.0
mg/mL in PBS containing 10% DMSO) was used for surface modification of islets
without further purification.
One hundred L urokinase (1 mg/mL in PBS) was mixed with 20 μL sulfoEMCS (7.6 μg in 20 μL PBS) for 2.5 hrs at RT to introduce maleimide groups onto
the enzyme surface. After purification with Sephadex G25, polyA-SH (28 μg, (2.0
mg/mL in PBS containing 10% DMSO)) was added to prepare polyA-urokinase.
The maleimide group of Mal-PEG-lipid was also used for the preparation of
PEG-modified islets for in vivo experiment. Its maleimide group was deactivated in
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advance by the addition of cystein as previously reported [11]. FITC-labeled PEGlipid [17] was used to visualize location of PEG-lipid on islets under a confocal laser
scanning microscopy (FLUOVIEW FV500, Olympus, Tokyo, Japan).
Surface modification of islets and their transplantation
Islets were isolated from the pancreases of BALB/c mice (6-7 weeks, females,
Japan SLC, Inc., Shizuoka, Japan) by the collagenase digestion method. Four mL of a
collagenase solution (0.5 mg/mL in HBSS) was injected into the common bile duct of
mice. The distended pancreas was removed and incubated at 37°C for 19 min. The
pancreatic tissue was disintegrated by pipetting. Islets were isolated from the
disintegrated tissue by the discontinuous gradient density fractionation using
Ficoll/Conray mixture solutions (density: 1.100, 1.075, 1.050). Tissue fragments
containing islets in high density were collected from the interface between
Ficoll/Conray 1.075 and Ficoll/Conray 1.050. Islets were picked up from the tissue
fragments by hand using a Pasteur pipette. The islets were cultured in Medium 199
with 10% FBS, 8.8 mM HEPES buffer, 100 units/mL penicillin, 100 g/mL
streptomycin, and 8.8 U/mL heparin for 2 days before surface modification.
A cystein-PEG-lipid solution (2.0 mg/mL in PBS containing 10% DMSO) was
added to suspended islets, and the mixture was incubated for 30 min at 37°C. After
washing with serum-free medium, cystein-PEG-lipid-modified islets (PEG-islets)
were obtained. They were transplanted into the livers of diabetic mice through the
portal vein.
Urokinase was immobilized to the surface through DNA hybridization
between polyA and polyT, as shown in Scheme 1b. A polyT-PEG-lipid solution (2
mg/mL in PBS containing 10% DMSO) was added to 100 L of islets suspended and
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the mixture was incubated for 30 min at 37°C. After washing with serum-free
medium,
polyA-PEG-lipid-modified
islets
(PEG-islets)
were
obtained.
For
preparation of urokinase-immobilized islets (urokinase-islets), polyA20-urokinase (30
μg in 30 μL PBS) was mixed with the polyT20-PEG-lipid modified islets and left for
1 h at RT for hybridization of polyT and polyA. After washing with serum-free
medium, urokinase-islets were transplanted into the livers of diabetic mice through
the portal vein.
In vitro glucose stimulation of modified islets
Static insulin secretion tests were performed on islets to evaluate their ability
to control insulin secretion in response to changes in glucose concentration. Fifty
islets were exposed to 1 mL of 0.1 g/dL, then 0.3 g/dL, and finally 0.1 g/dL of
glucose in Krebs-Ringer solution for 1 hour each at 37°C. The solutions were
collected after 1-hour incubation at different glucose concentration, and insulin
concentrations in these solutions were determined by ELISA.
Urokinase activity on islets
The fibrin plate assay was performed to examine fibrinolytic activities of
urokinase immobilized on islets, as previously reported [18,19]. Fibrin plates were
prepared as follows. Fibrinogen (type I, 80 mg) and plasminogen (0.2 mg) were
dissolved in saline (8 mL) and poured into a culture dish (ø =10 cm). Thrombin (60
IU/mL, 30 μL) was added to the fibrinogen solution and rotated. The plate was
incubated for 3 hr at RT to induce gelling of the solution. Urokinase immobilized
islets (100 islets) and bare islets (100 islets) were spotted on the fibrin plate. After
incubation at 37ºC for 16 hr, the fibrin plate was observed.
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Transplantation of islets into portal vein of STZ-induced diabetic mice
The Kyoto University Animal Care Committee of Institute of Frontier Medical
Sciences, Kyoto University approved all animal experiments. BALB/c mice (6 weeks,
males, Japan SLC, Inc. Shizuoka) were used as recipients. Diabetes was induced in
recipient mice by intraperitoneal injection of STZ (250 mg/kg body weight in citrate
buffer, pH 4.5) seven days prior to transplantation. An animal was considered diabetic
when its plasma glucose level exceeded 400 mg/dl on two consecutive measurements.
Naïve bare islets (125 and 250 islets), PEG-islets (125 islets), or urokinaseislets (125 islets) were transplanted into the liver through the portal vein of STZinduced diabetic mice, which were anesthetized during surgery by mask inhalation of
isoflurane using a specialized instrument (400 Anesthesia Unit; Univentor, Malta); the
isoflurane concentration was 4.5–5.0 % for induction and 2.0 % for maintenance with
an airflow rate of 200 mL/min. After transplantation, mice were housed in cages with
free access to food and water. Non-fasting plasma glucose levels were measured using
a glucose sensor (DIAmeter- glucocard; Arkray, Kyoto, Japan) before and after
transplantation. Blood samples were taken from the subclavian vein between 11 a.m.
and 1 p.m. Graft failure was defined as two consecutive plasma glucose levels over
200 mg/dL.
Intraperitoneal glucose tolerance test (IPGTT)
To evaluate glucose tolerance after transplantation of urokinase-islets and
PEG-islets, intraperitoneal glucose tolerance tests were performed at 64 days post
transplantation. Glucose solution (0.1 g/mL) was injected at a dose of 1.0 g/kg body
weight into the intraperitoneal cavity of mice that had been fasted for 6 h (9 a.m. to 3
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p.m.). Blood samples were taken before and after injection (0, 30, 60, and 120 min) to
determine plasma glucose levels. Glucose tolerance tests were also performed on
three normal Balb/c mice (7-weeks of age). Plasma glucose levels were determined by
a DIAmeter- glucocard.
Insulin levels in blood after transplantation
After transplantation of control islets, PEG-islets, or urokinase-islets (250
islets each, respectively) into liver of diabetic mice through the portal vein, blood was
taken from the subclavian vein at 15, 30, and 60 min and centrifuged to obtain
plasma. Insulin level in the plasma was determined by ELISA.
Histochemical analysis
Some recipient mice were sacrificed 64 days after transplantation for
histochemical examination. After livers were removed, blood was washed out by
perfusion of saline, and the tissue immersed in 10% formalin solution for 2 days at
RT. Paraffin embedding and preparation of tissue sections were performed by the
conventional method. The sections (4-μm thick) were permeabilized with 0.2% Triton
X-100 in PBS at RT for 15 min. For immunostaining of insulin, the sections were
then treated with 10% normal goat serum in PBS for 1 h to block non-specific binding
of antibodies and were then treated with 1% polyclonal guinea pig anti-insulin
antibody in PBS containing 3% goat normal serum for 3.5 hr at RT, followed by
washing with PBS. The sections were then incubated under a solution of fluorescent
labeled secondary antibody, 0.2% Alexa 488 Goat anti-guinea pig IgG in PBS
containing 3% goat normal serum at RT for 1.5 h. Cell nuclei were counterstained
with Hoechst 33342. The stained sections were examined under a fluorescence
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microscopy (BX51, Olympus Optical Co. Ltd., Tokyo, Japan). The sliced sections
were also counter-stained with hematoxylin-eosin (HE) using a conventional staining
method.
Statistical analyses
Comparisons between two groups were made using Student's t-tests. p<0.05
was considered statistically significant. All statistical calculations were performed
using the software KaleidaGraph 4.0J.
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