The effect of insulin-loaded linear poly(Ethylene glycol)-brush-like poly(L-lysine) block copolymer on renal ischemia/reperfusion-induced lung injury through downregulating hypoxia-inducible factor

Article abstract of DOI:10.2147/IJN.S99890

The aim of this study was to observe the therapeutic effect of insulin-loaded linear poly(ethylene glycol)-brush-like poly(l-lysine) block copolymer poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)) (PEG-b-(PELG-g-PLL) on renal ischemia/ reperfusion-induced lung injury through downregulating hypoxia-inducible factor (HIF) as compared to free insulin. Sprague Dawley rats were pretreated with 30 U/kg insulin or insulin/ PEG-b-(PELG-g-PLL) complex, and then subjected to 45 minutes of ischemia and 24 hours of reperfusion. The blood and lungs were collected, the level of serum creatinine and blood urea nitrogen were measured, and the dry/wet lung ratios, the activity of superoxide dismutase and myeloperoxidase, the content of methane dicarboxylic aldehyde and tumor necrosis factor-α, and the expression of HIF-1α and vascular endothelial growth factor (VEGF) were measured in pulmonary tissues. Both insulin and insulin/PEG-b-(PELG-g-PLL) preconditioning improved the recovery of renal function, reduced pulmonary oxidative stress injury, restrained inflammatory damage, and downregulated the expression of HIF-1α and VEGF as compared to ischemia/ reperfusion group, while insulin/PEG-b-(PELG-g-PLL) significantly improved this effect.

Full text of DOI:10.2147/IJN.S99890

International Journal of Nanomedicine
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Open ꢁccess FullText ꢁrticle
O ꢀ I g I N ꢁ ꢂ ꢀ ꢃ ꢄ ꢃ ꢁ ꢀ ꢅ h
Tꢆe effect of insulin-loaded linear poly(etꢆylene
ꢇlycol)-brusꢆ-like poly(l-lysine) block copolymer
on renal iscꢆemia/reperfusion-induced lunꢇ injury
tꢆrouꢇꢆ downreꢇulatinꢇ ꢆypoxia-inducible factor
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
2
7 April 2016
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Fei Tonꢇ
Xianꢇyuan Tanꢇ
Xin ꢂi
Abstract: The aim of this study was to observe the therapeutic effect of insulin-loaded linear
poly(ethylene glycol)-brush-like poly(l-lysine) block copolymer poly(ethylene glycol)-b-
(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)) (PEG-b-(PELG-g-PLL) on renal ischemia/
reperfusion-induced lung injury through downregulating hypoxia-inducible factor (HIF) as
compared to free insulin. Sprague Dawley rats were pretreated with 30 U/kg insulin or insulin/
PEG-b-(PELG-g-PLL) complex, and then subjected to 45 minutes of ischemia and 24 hours of
reperfusion. The blood and lungs were collected, the level of serum creatinine and blood urea
nitrogen were measured, and the dry/wet lung ratios, the activity of superoxide dismutase and
myeloperoxidase, the content of methane dicarboxylic aldehyde and tumor necrosis factor-α,
and the expression of HIF-1α and vascular endothelial growth factor (VEGF) were measured in
pulmonary tissues. Both insulin and insulin/PEG-b-(PELG-g-PLL) preconditioning improved the
recovery of renal function, reduced pulmonary oxidative stress injury, restrained inflammatory
damage, and downregulated the expression of HIF-1α and VEGF as compared to ischemia/
reperfusion group, while insulin/PEG-b-(PELG-g-PLL) significantly improved this effect.
Keywords: insulin, block copolymer, RI/RILI, HIF-1α, VEGF
Wenquan Xia
Daojun ꢂiu
Department of ꢅꢆemistry, Medical
ꢅolleꢇe, ꢄꢆantou University, ꢄꢆantou,
People’s ꢀepublic of ꢅꢆina
Introduction
1
Renal ischemia/reperfusion injury (RI/RI) is a major clinical problem. The morbidity
and mortality from RI/RI are very high and remain virtually unchanged for the past few
1,2
decades, in part because of a high incidence of extrarenal complications. In particular,
pulmonary damage is very frequent in patients suffering from RI/RI. It is a serious
complication of RI/RI. The extrarenal systemic complication secondary to RI/RI is
3
the leading cause of mortality in the intensive care unit. Indeed, clinical studies show
that patients with isolated RI/RI have significantly better prognosis than patients with
4
RI/RI plus extrarenal organ dysfunction.
Inflammation has a major function in the progression of RI/RI, and present studies
5,6
have shown that innate immunity contributes to the pathogenesis of RI/RI. Proinflam-
matory cytokines including tumor necrosis factor-α (TNF-α) and myeloperoxidase
(
MPO) generated by injured renal tissue have been implicated as the major contribu-
ꢅorrespondence: Daojun ꢂiu
Department of ꢅꢆemistry, Medical
ꢅolleꢇe, ꢄꢆantou University,
7
–9
tors of RI/RI. Indeed, increased circulating proinflammatory cytokine levels are
10
detected in rats and human beings with RI/RI. Induction of RI/RI results in increased
circulating proinflammatory cytokines (TNF-α and MPO), and these cytokines directly
cause pulmonary damage.
2
2 Xinlinꢇ ꢀoad, ꢄꢆantou 515041,
People’s ꢀepublic of ꢅꢆina
ꢃmail liudj@stu.edu.cn
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Hypoxia-inducible factor (HIF) is a heterodimeric basic
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Previous studies have demonstrated that HIF-1 is
implicated in most aspects of hypoxia-induced gene expression
and is essential for hypoxia-induced increases in glycolysis
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and angiogenesis in tumor cells as well as normal tissues.
High levels of HIF-1α expression and pronounced vascular
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beds with enhanced permeability are seen in tumor. It is par-
ticularly indicative of high levels of expression of one HIF-1
target, the angiogenic/vascular permeability factor vascular
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endothelial growth factor (VEGF). HIF-1α and VEGF are
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expressed in activated macrophages.
It has been reported
Figure 1 (A) ꢃncapsulation of insulin by linear-brusꢆ block copolymers. (B) Tꢆe
structure of Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ).
Note: m and n represent tꢆe deꢇree of polymerization.
Abbreviation: Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆy lenediamine
l-ꢇlutamate)-g-poly(l-lysine)).
that HIF-1α, its target gene VEGF, and its upstream regula-
tor, the von Hippel–Lindau factor, in separate mouse strains,
express cyclization recombination enzyme (Cre) recombinase
14
in granulocytes and monocytes/macrophages. Thisshowsthat
HIF-1αdirects regulation of survival and function in the inflam-
matorymicroenvironment. Renalischemiareperfusioncanlead
to pulmonary circulation dysfunction and can generate many
proinflammatory cytokines to lung and aggravate pulmonary
anoxia. These result in the activation of HIF to induce inflam-
matory responses in neutrophils and further damage the lung.
It has been demonstrated that insulin could protect the
isolated rat kidney from ischemia/reperfusion (I/R) injury
Materials and methods
ꢄyntꢆesis and cytotoxicity of block
copolymer Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
Detailed methods for the synthesis and cytotoxicity assay
of block copolymer PEG-b-(PELG-g-PLL) are supplied in
the Supplementary material.
ꢃncapsulation and in vitro release of
insulin in Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
Detailed methods for the encapsulation of insulin by PEG-b-
19
in diabetes mellitus. In the present study, we try to explore
the protective effect of insulin on RI/RI lung injury. As well
known, the in vivo half-life of insulin is very short; therefore,
controlling insulin dose clinically is very difficult with the risk
(
PELG-g-PLL) and in vitro release of insulin from PEG-b-
PELG-g-PLL) are supplied in the Supplementary material.
(
20
of hypoglycemia. Here, a linear poly(ethylene glycol) (PEG)-
brush-like poly(l-lysine)(PLL)blockcopolymerpoly(ethylene
glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly
ꢁ
nimal
Sprague Dawley rats (180–220 g) were supplied by the Animal
Research Center of Shantou University Medical College,
Shantou, People’s Republic of China. All the procedures
and care administered to the animals have been approved by
the Institutional Ethics Committee. In this study, 40 Sprague
Dawley rats were assigned to four groups (ten rats in each
group): 1) sham-operated group; 2) I/R group: I/R was
achieved by clamping renal artery and vein for 45 minutes fol-
lowed by 24 hours reperfusion; 3) Insulin + I/R group: insulin
was administrated once at a dose of 30 U/kg by abdominal
subcutaneous injection 6 hours prior to the I/R procedure;
(
l-lysine)) (PEG-b-(PELG-g-PLL) (structure shown in
Figure 1) as a potential insulin nanocarrier is reported.
The block copolymer is composed of a linear PEG block and
a brush-like PLL block. At physiological pH, the positively
charged PLL brush block and the negatively charged insulin
combine to form a polyion complex via electrostatic interac-
tions. The encapsulated insulin demonstrated sustained release,
which could facilitate the investigation on the protective effect
of insulin. Insulin-loading capacity and in vitro release were
determined. In vivo studies were conducted by the subcutane-
ous administration of the insulin/polymer complex to RI/RI
lung injury in rats, followed by the determination of serum
creatinine(Scr)andbloodureanitrogen(BUN), measurements
of superoxide dismutase (SOD) activity, methane dicarboxy-
4) Insulin/polymer + I/R group: insulin/polymer complexes
were administrated once at a dose of 30 U/kg by abdominal
subcutaneous injection 6 hours prior to the I/R procedure.
lic aldehyde (MDA), TNF-α and MPO level in pulmonary ꢄurꢇical procedures of I/ꢀ
tissues, and finally the expression of HIF-1α and VEGF in Rats were anesthetized with 1% pentobarbital sodium
pulmonary tissues.
(50 mg/kg) through intraperitoneal injection. The abdomen
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ꢃffect of insulin-loaded linear Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
was opened, and the renal artery and vein were separated. thawed three times. Homogenates were then centrifuged at
The artery and vein were clamped for 45 minutes by artery 10,000 rpm for 15 minutes at 4°C and the supernatant was
clamp followed by 24 hours of reperfusion. The blood collected for the measurement of MPO activity. The changes
samples were collected via abdominal aorta and centrifuged in absorbance at 460 nm were measured by spectrophotom-
at 3,600× g for 15 minutes to harvest the sera. A part of left etry and recorded at 20-second intervals for 10 minutes.
lung of rats was removed to weigh the wet lung, and then Results were expressed as U/μg of tissue.
baked for 48 hours at 80°C for weight of the dry lung. A part
of left lung was immediately excised and stored at -80°C for Western blot analysis
the following analyses.
Western blot analysis was conducted following the reported
2
5,26
methods.
Briefly, pulmonary tissues were homogenized
Measurement of serum BUN and ꢄcr
in protein lysate buffer. The homogenates were resolved
Serum BUN and Scr were measured according to the reported on polyacrylamide sodium dodecyl sulfate gels and elec-
2
1
methods. Briefly, the blood from abdominal aorta was trophoretically transferred to polyvinylidene difluoride
collected after reperfusion for 24 hours and centrifuged at membranes. The membranes were blocked with 3% bovine
3
,600× g for 15 minutes to harvest the sera. The serum BUN serum albumin, incubated with primary antibodies against
and Scr were measured by o-phthalaldehyde and picric acid active HIF-1α and VEGF, and subsequently with alkaline
2
1
methods, respectively.
phosphatase-conjugated secondary antibodies. HIF-1α and
VEGF were developed by 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium. Blots were stained with
anti-β-actin Ab, and the levels of proteins were normalized
Measurement of ꢄOD activity and MDꢁ
content
The levels of SOD and MDA in tissues were measured with respect to β-actin band density.
22
following the reported methods. The lung was collected
after reperfusion for 24 hours, and the blood was washed ꢄtatistical analysis
thoroughly, homogenized (100 mg) in ice-cold normal All data were expressed as mean values ± standard deviation.
saline, and then centrifuged at 3,600× g for 15 minutes to Statistical analysis between groups was carried out using
harvest the supernatant. The SOD activity and MDA content analysis of variance with post hoc testing. A P-value of less
were measured by xanthine oxidase and thiobarbituric acid than 0.01 (P,0.01) was used for statistical significance.
2
2
method. The absorbance was measured at 550 and 532 nm,
respectively. The level of lipid peroxides was expressed as U
of SOD/mg protein and nmol of MDA/mg tissue.
Results
ꢄyntꢆesis and cꢆaracterization of Pꢃg-b-
(
Pꢃꢂg-g-Pꢂꢂ)
Measurement ofTNF-α content
The linear poly(ethylene glycol)-brush-like poly(l-lysine)
The content of TNF-α in pulmonary tissues was determined block copolymer PEG-b-(PELG-g-PLL) consists of a linear
2
3
according to the reported method. Lung tissue stored PEG block and a brush-like PLL block (Figure 1A and B).
at -80°C was thawed and homogenized in a buffer composed In the present study, the molecular weight of PEG block was
of phosphate-buffered saline, 0.5% Triton X-100, and pro- 5 kDa, the polymerization degree of the PELG brush back-
tease inhibitor, pH 7.2. The material was then centrifuged bone was 50, while the polymerization degree of the PLL
at 3,600 rpm for 15 minutes at 4°C and the supernatant was brush side chains was 3. The synthesis was performed in a
1
collected for enzyme-linked immunosorbent assay. TNF-α four-step procedure as shown in Figure 2. The H-nuclear
level in lung tissue was determined by immunoassay in magnetic resonance (NMR) spectra of PEG-b-(PELG-g-
accordance with the manufacturer’s directions. Results were PLL) and its intermediates are shown in Figure 3. As seen
expressed in pg/mL.
in Figure 3A, the characteristic proton peaks of both PEG
and poly(benzyl-l-glutamate) (PBLG) block were present,
confirming that the synthesis proceeded in a controlled
Measurement of MPO activity
The MPO activity of pulmonary tissues was evaluated fol- manner. Characteristic peaks of ethylenediamine are seen in
24
lowing the reported method. MPO extraction was performed Figure 3B, indicating the success of the aminolysis reaction.
1
by homogenization of the lung tissue in phosphate buffer, pH Figure 3C demonstrates the H NMR spectrum of PEG-b-
1
6
.0, containing 0.5% hexadecyltrimethylammonium bromide. (PELG-g-PZLL) in DMSO-d , while the H NMR spectrum
6
The samples were sonicated, frozen in liquid nitrogen, and of the final block copolymer PEG-b-(PELG-g-PLL) in D O
2
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Tonꢇ et al
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O
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PEG-b-(PELG-g-PLL)
Figure 2 ꢄyntꢆesis of linear-brusꢆ copolymer of Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ).
Abbreviations: Bꢂg, γ-benzyl l-ꢇlutamate;BTꢅ, bis(tricꢆlorometꢆyl)carbonate;Bꢂg-Nꢅꢁ, γ-benzyl l-ꢇlutamate-N-carboxyanꢆydride;Pꢃg-Nh :poly(etꢆylene ꢇlycol) amine;
2
Pꢃg-b-PBꢂg, poly(etꢆylene ꢇlycol)-b-poly(γ-benzyl l-ꢇlutamate); DMF, N, N-dimetꢆylformamide; 2-hP, 2-ꢆydroxypyridine; ꢃꢁ, etꢆylenediamine; Pꢃg-b-Pꢃꢂg, poly(etꢆylene
ꢇlycol)-b-poly(etꢆylenediaminel-ꢇlutamate);Zꢂꢂ-Nꢅꢁ,ε-benzyoxycarbonyl-l-lysine-N-carboxyanꢆydride;Pꢃg-b-(Pꢃꢂg-ꢇ-PZꢂꢂ),poly(etꢆyleneꢇlycol)-b-(poly(etꢆylenediamine
l-ꢇlutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)); Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆylenediamine l-ꢇlutamate)-g-poly(l-lysine)).
is shown in Figure 3D. The disappearance of proton peak of and the results are shown in Figure 5A. The block copolymer
benzyl groups in Figure 3D suggests that PEG-b-(PELG-g- exhibited high cell viability even under a concentration as
PLL) was synthesized successfully.
high as 500 mg/L.
The gel permeation chromatograms (GPCs) of PEG-b-
(
PELG-g-PLL) and its intermediates are shown in Figure 4,
ꢂoadinꢇ capacity of insulin in Pꢃg-b-
and the results are listed in Table 1. All GPCs demonstrated
a sharp unimodal GPC peak, which suggests that the block
copolymer is synthesized in a controlled manner. The
molecular weight of the final product PEG-b-(PELG-g-PLL)
obtained from the GPC measurement (20.8 kDa) was lower
(
Pꢃꢂg-g-Pꢂꢂ)
Insulin can be efficiently entrapped by block copolymer
PEG-b-(PELG-g-PLL) under physiological pH via the elec-
trostatic interactions between the positively charged PLL and
the negatively charged insulin. Fluorescein isothiocyanate-
labeled insulin was mixed with PEG-b-(PELG-g-PLL) at
a mass ratio of 1:5 and dialyzed (molecular weight cut off
1
than the calculated value from H NMR (31.3 kDa), possi-
bly because of the smaller hydrodynamic volumes of brush
polymers compared with linear polymers.
[
(
MWCO], 100 kDa) against phosphate buffer (PB) solution
pH 7.4, 0.01 mmol/L). The dialysis of free insulin in a PB
ꢅell viability
solution was also conducted under the same conditions as a
The cytotoxicity of block copolymer PEG-b-(PELG-g-PLL) control. To determine the loading capacity of insulin in PEG-
on EC9706 cells was monitored within 24 hours of culturing, b-(PELG-g-PLL), the amount of insulin in the dialysate was
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ꢃffect of insulin-loaded linear Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
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Notes: (A) Pꢃg-b-PBꢂg in ꢅDꢅl ; (B) Pꢃg-b-Pꢃꢂg in D O; (C) Pꢃg-b-(Pꢃꢂg-g-PZꢂꢂ) in DMꢄO-d ; (D) Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ) in D O. Peak labels (a–k) in eacꢆ spectrum
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Abbreviations:
h NMꢀ, h-nuclear maꢇnetic resonance; Pꢃg-b-PBꢂg, poly(etꢆylene ꢇlycol)-b-poly(γ-benzyl l-ꢇlutamate); Pꢃg-b-Pꢃꢂg, poly(etꢆylene ꢇlycol)-b-
poly(etꢆylenediamine l-ꢇlutamate); Pꢃg-b-(Pꢃꢂg-g-PZꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆylenediamine l-ꢇlutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)); Pꢃg-b-(Pꢃꢂg-g-
Pꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆylenediamine l-ꢇlutamate)-g-poly(l-lysine)); DMꢄO, dimetꢆylsulfoxide.
measured using a fluorimeter and then subtracted from the 46 nm, respectively. The larger diameter observed by trans-
total amount of added insulin. The loading capacity of insulin mission electron microscope might be due to the aggregation
was determined to be 11.5%, expressed as the mass ratio of of complexes during the drying process.
encapsulated insulin to the polymer host (Table 1).
In vitro release of insulin from Pꢃg-b-
ꢅꢆaracterization of insulin/polymer
complex
(
Pꢃꢂg-g-Pꢂꢂ)
The release of insulin from PEG-b-(PELG-g-PLL) was
The insulin/polymer complex was visualized with transmis- investigated using a dialysis method (MWCO, 100 kDa)
sion electron microscope, and the representative image is at room temperature, with 5 mL of insulin-loaded polymer
shown in Figure 5B. The insulin/polymer complex adopted against a PB solution of 0.02 mol/L (pH of 7.4). The cumula-
a spherical structure, and the diameter was approximately tive release profile of polymer-encapsulated insulin is shown
9
0 nm. The insulin/polymer complex was further character- in Figure 5E. After 2 hours, approximately 32.7% of the
ized with a granulometer. The average particle size distribu- insulin was released from insulin/polymer, indicative of an
tions of free polymer and insulin-loaded polymer are shown initial burst release of insulin. Approximately 57% of the
in Figure 5C and D, and Table 1. The average diameters of insulin was released after 3 days and approximately 70%
block copolymer PEG-b-(PELG-g-PLL) and insulin/polymer after 5 days, indicative of biphase release profiles from
complex (10% insulin loading) were approximately 13 and insulin/polymer.
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Tonꢇ et al
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A
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Retention time (min)
Retention time (min)
Figure 4 gPꢅs of (A) Pꢃg-b-PBꢂg; (B) Pꢃg-b-Pꢃꢂg; (C) Pꢃg-b-(Pꢃꢂg-g-PZꢂꢂ); and (D) Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ).
Abbreviations: gPꢅ, ꢇel permeation cꢆromatoꢇram; Pꢃg-b-PBꢂg, poly(etꢆylene ꢇlycol)-b-poly(γ-benzyl l-ꢇlutamate); Pꢃg-b-Pꢃꢂg, poly(etꢆylene ꢇlycol)-b-
poly(etꢆylenediamine l-ꢇlutamate); Pꢃg-b-(Pꢃꢂg-g-PZꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆylenediamine l-ꢇlutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)); Pꢃg-b-(Pꢃꢂg-
g-Pꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆylenediamine l-ꢇlutamate)-g-poly(l-lysine)).
in I/R group rats. Administration of insulin reduced the
ꢄerum level of BUN and ꢄcr
level of Scr (18.01±3.08 μmol), whereas administration
of insulin/polymer significantly reduced the level of Scr
As shown in Figure 6A and B, the serum levels of BUN and
Scr in I/R group rats were higher than that in sham group
rats (P,0.01). The level of BUN was 6.05±1.73 mmol
in sham group rats, whereas the level of BUN reached
(14.48±2.78 μmol) compared with I/R group rats (P,0.01).
20.85±2.03 mmol in I/R group rats. Administration of insulin Dry/wet lunꢇ ratios
reduced the levels of BUN (17.51±2.18 mmol), whereas As shown in Figure 7, the dry/wet (D/W) lung ratios in I/R
administration of insulin/polymer significantly reduced the group rats were less than that in the sham group (P,0.01).
levels of BUN (13.03±1.98 mmol) compared with I/R group The D/W lung ratio reached 27.5±1.08 in the sham group,
rats (P,0.01). The level of Scr was 9.16±3.18 μmol in sham whereas the D/W lung ratio in I/R group reached 16.6±0.645.
group rats, whereas the level of Scr reached 22.49±4.23 μmol Administration of insulin increased the D/W lung ratios
Table 1 Molecular weiꢇꢆts, polydispersities, particle size, TꢃM, and insulin-loadinꢇ capacity of Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
1
Sample
M (kDa)/ H NMR
M (kDa)/GPC
PDI
Particle size (nm)
TEM (nm)
Loading capacity (%)
n
n
Polymer
Insulin/polymer
31.3
Nꢁ
20.8
Nꢁ
2.07
Nꢁ
13
46
Nꢁ
90
Nꢁ
11.5
1
1
Abbreviations: TꢃM, transmission electron microscopy; h NMꢀ, h-nuclear maꢇnetic resonance; gPꢅ, ꢇel permeation cꢆromatoꢇrapꢆy; PDI, polydispersity index;
M , number-averaꢇe molecular weiꢇꢆt; Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆylenediamine l-ꢇlutamate)-g-poly(l-lysine)); Nꢁ, not applicable.
n
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Figure 5 ꢅꢆaracterization of Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ) and insulin/Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ) complexes.
Notes: (A) ꢅellular viability of ꢃꢅ9706 cells cultured witꢆ different concentrations of Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ); (B) TꢃM imaꢇe of insulin/Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ) complexes;
C) Diameter of block copolymer Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ) in PB; (D) Diameter of insulin/Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ) complexes in PB; (E) Cumulative releasing profile of insulin from
(
insulin/Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ) complexes.
Abbreviations: TꢃM, transmission electron microscopy; PB, pꢆospꢆate buffer; Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ), poly(etꢆylene ꢇlycol)-b-(poly(etꢆylenediamine l-ꢇlutamate)-g-poly(l-lysine)).
(20.0±1.53) compared with the D/W lung ratio in I/R group group (P,0.01). The activity of SOD in pulmonary tissues
rats (P,0.01), whereas administration of insulin/polymer sig- reached 130.1±2.15 U in sham group, whereas the activity of
nificantly increased the D/W lung ratio (24.0±1.46), whichwas SOD in pulmonary tissues in I/R group reached 67.23±1.18 U.
significantly greater than that in the I/R groups (P,0.01).
Administration of insulin increased the activity of SOD
(
83.86±2.35 U) in pulmonary tissues, compared with the
Tꢆe activity of ꢄOD and tꢆe level of MDꢁ
in pulmonary tissues
activity of SOD in pulmonary tissues in the I/R group rats
(P,0.01). Administration of insulin/polymer significantly
As shown in Figure 8A and B, the activity of SOD in pul- increased the activity of SOD (103.1±1.79 U), which was
monary tissues in I/R group rats is less than that in the sham significantly greater than that in the I/R groups (P,0.01).
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Tonꢇ et al
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Figure 6 ꢂevels of serum BUN (A) and ꢄcr (B).
Notes: Tꢆe serum of ꢄꢆam, I/ꢀ, Insulin, and Insulin/polymer ꢇroups rats was collected 24 ꢆours after reperfusion and results expressed as mean ± ꢄD. (A) A significant
increase from ꢄꢆam ꢇroup is denoted by *P,0.01, a significant decrease from I/R groups is denoted by **P,0.01, and a significant decrease from I/R groups is denoted by
*
**P,0.01. (B) A significant increase from Sham group is denoted by *P,0.01, a significant decrease from I/R groups is denoted by **P,0.01, and a significant decrease from
I/ꢀ ꢇroups is denoted by ***P,0.01.
Abbreviations: BUN, blood urea nitroꢇen; ꢄcr, serum creatinine; I/ꢀ, iscꢆemia/reperfusion; ꢄD, standard deviation.
The level of MDA in pulmonary tissues in I/R group rats
Tꢆe level ofTNF-α and tꢆe activity of MPO
was higher than that in the sham group (P,0.01). The level
of MDA in pulmonary tissues reached 1.28±0.34 nmol in
sham group, whereas the level of MDA in pulmonary tis-
sues in the I/R group reached 2.17±0.49 nmol. Administra-
tion of insulin reduced the level of MDA (1.94±0.41 nmol)
in pulmonary tissues, compared with the level of MDA in
pulmonary tissues in the I/R group rats (P,0.01). Adminis-
tration of insulin/polymer significantly reduced the level of
MDA (1.52±0.29 nmol), which was significantly less than
that in the I/R groups (P,0.01).
in pulmonary tissues
As shown Figure 9A and B, the level of TNF-α in pulmonary
tissues in I/R group rats was higher than that in the sham
group (P,0.01). The level of TNF-α in pulmonary tissues
reached 10.17±1.89 pg in the sham group, whereas the level
of TNF-α in pulmonary tissues reached 39.15±2.35 pg in the
I/R group. Administration of insulin reduced the level of
TNF-α (30.65±2.09 pg) in pulmonary tissues, compared with
the level of TNF-α in pulmonary tissues in the I/R group rats
(P,0.01). Administration of insulin/polymer significantly
reduced the level of TNF-α (26.27±2.13 pg), which was
significantly less than that in the I/R groups (P,0.01).
The activity of MPO in pulmonary tissues in I/R group
rats was higher than that in the sham group (P,0.01). The
activity of MPO in pulmonary tissues reached 8.32±2.19 U
in the sham group, whereas the activity of MPO in pulmonary
tissues in the I/R group reached 33.17±3.15 U. Administra-
tion of insulin decreased the activity of MPO (28.32±2.03 U)
in pulmonary tissues, compared with the activity of MPO in
pulmonary tissues in I/R group rats (P,0.01). Administra-
tion of insulin/polymer significantly decreased the activity
of MPO (22.16±1.98 U), which was significantly less than
that in the I/R groups (P,0.01).
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Figure 7 Tꢆe level of D/W lunꢇ ratios for different ꢇroups of rats.
in pulmonary tissues
Notes: Tꢆe pulmonary tissues of ꢄꢆam, I/ꢀ, Insulin, and Insulin/polymer ꢇroups
rats were collected 24 ꢆours after reperfusion and tꢆe levels of D/W lunꢇ ratios
measured. ꢀesults are expressed as mean ± SD. A significant decrease from
ꢄꢆam ꢇroup is denoted by *P,0.01, a significant increase from I/R groups is
denoted by **P,0.01, and a significant increase from I/R groups is denoted by
HIF-1α and VEGF expression in pulmonary tissues was
measured by Western blot following the reported method.
25,26
The results were analyzed both qualitatively and quantita-
*
**P,0.01.
Abbreviations: D/W, dry/wet; I/ꢀ, iscꢆemia/reperfusion; ꢄD, standard deviation.
tively (Figure 10). The expression of HIF-1α in pulmonary
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Dovepress
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Notes: Tꢆe pulmonary tissues of ꢄꢆam, I/ꢀ, Insulin, and Insulin/polymer ꢇroups rats were collected 24 ꢆours after reperfusion and tꢆe activity of ꢄOD and tꢆe level of MDꢁ
measured. ꢀesults are expressed as mean ± ꢄD. (A) A significant decrease from Sham group was denoted by *P,0.01, a significant increase from I/R groups, by **P,0.01,
and a significant increase from I/R groups, by ***P,0.01; (B) A significant increase from sham group was denoted by *P,0.01, a significant decrease from I/R groups, by
*
*P,0.01, and a significant decrease from I/R groups, by ***P,0.01.
Abbreviations: ꢄOD, superoxide dismutase; I/ꢀ, iscꢆemia/reperfusion; MDꢁ, metꢆane dicarboxylic aldeꢆyde; ꢄD, standard deviation.
tissues in I/R groups was higher than that in the sham group expression of VEGF, compared with that in the I/R group
(
P,0.01). Administration of insulin downregulated the (P,0.01).
expression of HIF-1α in pulmonary tissues, compared with
the expression of HIF-1α by I/R group (P,0.01). Adminis- Discussion
tration of insulin/polymer complex significantly downregu- This is the first study to demonstrate that insulin and insulin/
lated the expression of HIF-1α, compared with that in the PEG-b-(PELG-g-PLL) preconditioning are both capable
I/R group (P,0.01).
of attenuating remote lung injury induced by renal I/R.
The expression of VEGF in pulmonary tissues in I/R It was found that pulmonary inflammatory response was
groups was higher than that in the sham group (P,0.01). significantly relieved after insulin and insulin/PEG-b-(PELG-
Administration of insulin downregulated the expression of g-PLL) pretreatment. This protective effect is partially down-
VEGF in pulmonary tissues, compared with the expression regulated with HIF, indicating that the underlying mechanism
of VEGF in the I/R group (P,0.01). Administration of is associated with either HIF-dependent or -independent
insulin/polymer complex significantly downregulated the pathways. In the present study, the blood glucose levels
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Notes: Tꢆe pulmonary tissues of ꢄꢆam, I/ꢀ, Insulin and Insulin/polymer ꢇroups rats were collected 24 ꢆours after reperfusion and tꢆe level of TNF-α and tꢆe activity of
MPO measured. ꢀesults are expressed as mean ± ꢄD. (A) A significant increase from sham group is denoted by *P,0.01, a significant decrease from I/R groups is denoted
by **P,0.01, and a significant decrease from I/R groups is denoted by ***P,0.01. (B) A significant increase from sham group is denoted by *P,0.01, a significant decrease
from I/ꢀ ꢇroups is denoted by **P,0.01, and a significant decrease from I/R groups is denoted by ***P,0.01.
Abbreviations: TNF, tumor necrosis factor; I/ꢀ, iscꢆemia/reperfusion; MPO, myeloperoxidase; ꢄD, standard deviation.
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Figure 10 Tꢆe qualitative expression (C) and quantitative analyses of hIF-1α (A) and VꢃgF (B) in pulmonary tissues for different ꢇroups of rats.
Notes: Tꢆe pulmonary tissues of ꢄꢆam, I/ꢀ, Insulin, and Insulin/polymer ꢇroups rats were collected 24 ꢆours after reperfusion and tꢆe expression of hIF-1α and VꢃgF
measured. ꢀesults are expressed as mean ± ꢄD. (A) A significant increase from sham group is denoted by *P,0.01, a significant decrease from I/R groups is denoted by
*
*P,0.01, and a significant decrease from I/R groups is denoted by ***P,0.01. (B) A significant increase from sham group is denoted by *P,0.01, a significant decrease from
I/ꢀ ꢇroups is denoted by **P,0.01, and a significant decrease from I/R groups is denoted by ***P,0.01.
Abbreviations: hIF, ꢆypoxia-inducible factor; VꢃgF, vascular endotꢆelial ꢇrowtꢆ factor; I/ꢀ, iscꢆemia/reperfusion; ꢄD, standard deviation.
were traced after insulin or insulin/polymer complexes pretreatment can reduce the expression of HIF-1α and TNF-α.
were injected; it should be noted that hypoglycemia was not In addition, TNF-α can increase lung MPO activity after
27
37
observed, in accordance with reported literature.
acute kidney injury. MPO is released from neutrophils and
RI/RI occurs in approximately 5% of hospitalized patients catalyzes the formation of hypochlorous acid from hydrogen
28,29
37
and up to 30% of patients in intensive care units.
Pre- peroxide and chloride ions. The MPO level indirectly reflects
37
treatment with insulin can reduce the injury for hospitalized the severity of inflammatory response. Our study suggests
patients. At present, insulin preconditioning has been used that insulin and insulin/PEG-b-(PELG-g-PLL) pretreatment
clinically in many hospitals to reduce ischemia reperfusion can inhibit the activation of HIF-1α to reduce the activity of
30
injury. Systemic inflammation following renal ischemia/ MPO in pulmonary tissues.
3
8
reperfusion (RI/R) can progress into multiple organ damage VEGF is an important vascular permeability factor.
31
if inflammatory response is unchecked. Indeed, RI/RI causes High expression of VEGF within the lung may contribute
upregulation of inflammatory mediators in local and remote to the development of pulmonary edema by deteriorating the
32
organs. The release and migration of inflammatory factors state of the adherens junction complexes on the endothelium
33
need a lot of adenosine triphosphate (ATP). Inflammatory and aggravate microcirculation disturbance and inflamma-
34–36
33,39
cells depend principally on glycolysis for ATP generation.
tory response.
Our study shows that insulin and insulin/
HIF-1α is one of the most important nuclear factors that PEG-b-(PELG-g-PLL) pretreatment can reduce the expres-
adjusts the expression of anaerobic glycolysis key enzyme in sion of VEGF through downregulating HIF-1α to decrease
33
inflammatory cells. The present study shows that inhibition the inflammatory response of pulmonary tissues.
of HIF-1α activation can reduce anaerobic glycolysis ability Insulin pretreatment can protect pulmonary tissue against
in inflammatory cells and ATP content in cells to weaken renal ischemia/reperfusion-induced injury. However, insu-
the adhesion, chemotaxis, and proinflammatory ability of lin has a short half-life and its dose is difficult to control,
14
inflammatory cells. Our study clearly demonstrates that RI/R and frequent insulin injection increases the suffering of the
2
0
rapidly leads to distant overexpression of HIF-1α and TNF-α patient. In our report, block copolymer PEG-b-(PELG-
in the lungs. Insulin and insulin/PEG-b-(PELG-g-PLL) g-PLL) serves as a carrier and protects insulin from rapid
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ꢃffect of insulin-loaded linear Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
3
4
. Faubel S. Pulmonary complications after acute kidney injury. Adv
Chronic Kidney Dis. 2008;15:284–296.
degradation in the body and can reinforce efficacy, and reduce
dosage and side effects. In addition, PEG-b-(PELG-g-PLL)
can increase the stability of insulin in circulation. This study
reveals that PEG-b-(PELG-g-PLL) treatment significantly
protects lung against RI/R-induced injury in rats, compared
to free insulin. In summary, our current study shows that
insulin and insulin/PEG-b-(PELG-g-PLL) pretreatment can
provide lung protection after RI/R-induced injury through
downregulating HIF, whereas insulin/PEG-b-(PELG-g-PLL)
significantly improves this effect.
. Bagshaw SM, Laupland KB, Doig CJ, et al. Prognosis for long-term sur-
vival and renal recovery in critically ill patients with severe acute renal
failure: a population-based study. Crit Care. 2005;9:R700–R709.
. Burne-Taney MJ, Rabb H. The role of adhesion molecules and T cells in
ischemic renal injury. Curr Opin Nephrol Hypertens. 2003;12:85–90.
. Rabb H. Immune modulation of acute kidney injury. J Am Soc Nephrol.
5
6
7
2
006;17:604–606.
. Donnahoo KK, Meldrum DR, Shenkar R, Chung CS, Abraham E,
Harken AH. Early renal ischemia, with or without reperfusion, activates
NF-κB and increases TNF-α bioactivity in the kidney. J Urol. 2000;163:
1328–1332.
8
. Donnahoo KK, Meng X, Ayala A, Cain MP, Harken AH, Meldrum DR.
Early kidney TNF-α expression mediates neutrophil infiltration
and injury after renal ischemia-reperfusion. Am J Physiol. 1999;
Conclusion
2
77:R922–R929.
The synthesized block copolymer PEG-b-(PELG-g-PLL)
can significantly improve the bioactivity of insulin. The
observed protective effect can be attributed to the antioxida-
tive stress and the modulation of HIF activation by insulin/
PEG-b-(PELG-g-PLL) in rats as compared to free insulin.
Administration of insulin or insulin/PEG-b-(PELG-g-PLL)
demonstrated protective effect against RI/R-induced pulmo-
nary injury through improving renal function and decreasing
the level of MDA, TNF-α, the activity of MPO, downregulat-
ing the expression of HIF-1α and VEGF, and increasing the
activity of SOD, while significantly improving the protec-
tive effect. More detailed studies are required to explore the
underlying mechanism of insulin/PEG-b-(PELG-g-PLL) on
RI/R-induced pulmonary injury in rats.
9. Oztay F, Kara-Kisla B, Orhan N, Yanardag R, Bolkent S. The protective
effects of prostaglandin E1 on lung injury following renal ischemia-
reperfusion in rats. Toxicol Ind Health. Epub April 16, 2015.
10. Liu KD, Altmann C, Smits G, et al. Serum interleukin-6 and
interleukin-8 are early biomarkers of acute kidney injury and predict
prolonged mechanical ventilation in children undergoing cardiac
surgery: a case control study. Crit Care. 2009;13:R104.
11. Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-α to the von
Hippel-Lindau ubiquitylation complex by O regulated prolyl hydroxy-
2
lation. Science. 2001;292:468–472.
12. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1
is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2
tension. Proc Natl Acad Sci U S A. 1995;92:5510–5514.
1
1
3. Semenza GL. Hypoxia-inducible factor 1: control of oxygen homeo-
stasis in health and disease. Pediatr Res. 2001;49:614–617.
4. Cramer T, Yamanishi Y, Clausen BE, et al. HIF-1α is essential for
myeloid cell-mediated inflammation. Cell. 2003;112(5):645–657.
15. Maxwell PH, Wiesener MS, Chang GW, et al. The tumor suppressor
protein VHL targets hypoxia-inducible factors for oxygen-dependent
proteolysis. Nature. 1999;399:271–275.
1
6. Burke B, Tang N, Corke KP, et al. Expression of HIF-1α by human
macrophages: implications for the use of macrophages in hypoxia-
regulated cancer gene therapy. J Pathol. 2002;196:204–212.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (50973058) and the Department
of Education, Guangdong Government under the Top-tier
University Development Scheme for Research and Control
of Infectious Diseases.
1
7. Hollander AP, Corke KP, Freemont AJ, Lewis CE. Expression of
hypoxia-inducible factor 1α by macrophages in the rheumatoid syn-
ovium: implications for targeting of therapeutic genes to the inflamed
joint. Arthritis Rheum. 2001;44:1540–1544.
8. Talks KL, Turley H, Gatter KC, et al. The expression and distribution
of the hypoxia-inducible factors HIF-1α and HIF-2α in normal human
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Author contributions
19. Melin J, Hellberg O, Larsson E, Zezina L, Fellstrom BC. Protective effect
of insulin on ischemia renal injury in diabetes mellitus. Kidney Int. 2002;
All authors contributed toward data analysis, drafting and
critically revising the paper, gave final approval of the ver-
sion to be published, and agree to be accountable for all
aspects of the work.
6
1:1383–1392.
20. InceBW.Plasmaclearancekineticsofunlabeledbovineinsulininrainbow
trout (Salmo gairdneri). Gen Comp Endocrinol. 1982;46:463–472.
2
1. Li Z, Wang Y. Effect of NADPH oxidase inhibitor-apocynin on the
expressionofSrchomology-2domain-containingphosphatase-1(SHP-1)
exposed renal ischemia/reperfusion injury in rats. Toxicology Rep. 2015;
2
:1111–1116.
Disclosure
The authors report no conflicts of interest in this work.
2
2. Jiang Y, Zhou Z, Meng QT, et al. Ginsenoside Rb1 treatment attenuates
pulmonary inflammatory cytokine release and tissue injury following
intestinal ischemia reperfusion injury in mice. Oxid Med Cell Longev.
2
015;2015:843721.
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ꢃffect of insulin-loaded linear Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
Supplementary materials
2-hydroxypyridine, and 20 times the amount of ethylene-
diamine was added by vacuumizing and N protection. The
Materials
2
o
reaction mixture was stirred at 40 C for 2 days and then 10%
Amino polyethylene glycol monomethyl ether (PEG-NH ,
2
acetic acid solution was slowly added and stirred for 3 hours
while immersed in an ice bath, then dialyzed for 3 days
molecular weight =5 kDa), 2-hydroxypyridine, trifluo-
roacetic acid, dimethyl formamide (DMF), ethylacetate, and
petroleumether were purchased from Aladdin (Shanghai,
People’s Republic of China); Bis(trichlormethyl)carbonate,
ethylenediamine, and hydrogen bromide/acetic acid solution
(
MWCO, 3.5 kDa). The PEG-b-PELG was obtained by freeze
1
drying, and characterized by H NMR, GPC and FT-IR.
ZLL-NCA was synthesized by the phosgenation of ZLL
in anhydrous ethyl acetate following previously reported
(
HBr/CH COOH, 33%) were purchased from Alfa (Tian,
3
2
,3
1
methods and characterized by H NMR. The proper
amounts of PEG-b-PELG in DMF (0.1 g/3.5 mL) were
mixed with a corresponding amount of ZLL-NCA/DMF
People’s Republic of China); γ-benzyl l-glutamate (BLG)
and ε-benzyoxycarbonyl–l–lysine (ZLL) were purchased
from Shanghai Jill biochemical co., LTD, (Shanghai, People’s
Republic of China). Regenerated cellulose membrane tubing
solution (0.1 g/mL) by vacuumizing and N protection. The
2
o
reaction mixture was stirred at 40 C for 3 days and dialyzed
(molecular weight cut-off [MWCO], 3.5 kDa, 7 kDa) were
for 3 days (MWCO, 7 kDa). The PEG-b-(PELG-g-PZLL)
purchased from Union Cardibide Company (Danbury, CT,
USA); Insulin and other reagent are from Sigma-Aldrich
was obtained by freeze drying, and the degree of polym-
1
erization of PZLL was 3, and characterized by H NMR,
(St Louis, MO, USA).
GPC and FT-IR.
Metꢆods
ꢄyntꢆesis of poly(etꢆylene ꢇlycol)-b-
Deprotection of the benzyl groups in PEG-b-(PELG-g-
4
PZLL) was performed following the reported method. The
(
(
poly(etꢆylenediamine l-ꢇlutamate)-g-poly(l-lysine))
Pꢃg-b-(Pꢃꢂg-g-Pꢂꢂ)
proper amounts of PEG-b-(PELG-g-PZLL) were mixed for a
short duration with the corresponding amount of CF COOH
3
PEG-b-(PELG-g-PLL) was synthesized in the following and HBr/CH COOH was slowly added and allowed to
3
method: PEG-b-PBLG was prepared by the ring-opening react for 1 hour. The product was isolated by precipitation
polymerization of BLG-N-carboxyanhydride (NCA) with in diethyl ether and filtration, followed by neutralization
PEG-NH as the macroinitiator, followed by aminolysis with with NaHCO aqueous solution. The solution was dialyzed
2
3
ethanediamine to obtain PEG-b-PELG. PEG-b-PELG was for 5 days (MWCO, 3.5 kDa) and the PEG-b-(PELG -
5
0
used as the macroinitiator to initiate the ring-opening polymer- g-PLL ) polymer was obtained by freeze drying, and was
3
1
ization of ZLL-NCA to prepare PEG-b-(PELG-g-PZLL). The characterized by H NMR, GPC and FT-IR.
benzyl groups of poly(ε-benzyloxycarbonyl-l-lysine) were
then deprotected in the presence of HBr to obtain the final ꢅytotoxicity assay of polymers
5
product, PEG-b-(PELG-g-PLL), as depicted in Figure 2.
EC9706 cells were used to assess biomaterial cytotoxicity.
First, BLG-NCA was synthesized by the phosgenation We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
of BLG in anhydrous ethyl acetate, following the reported zolium bromide (MTT) colorimetric assay, as described by
1
1
6
method of Hideharu et al and characterized by H nuclear Mosmann. The cells were cultured in a 96-well plate with
3
magnetic resonance (NMR). BLG-NCA was polymerized 5×10 cells per well. Twenty-four hours after cell incubation,
in DMF using PEG-NH as the macroinitiator. The proper the original medium was removed and MTT solution was
2
amounts of PEG-NH in 10 mL of DMF were added to a cor- added to each well. Only, the cells that adhered to the well
2
responding amount of BLG-NCA/DMF solution (0.1 g/mL) walls were incubated with MTT for 24 hours at 37°C. During
by vacuumizing and N protection. The reaction mixture was incubation, the MTT was reduced by mitochondrial succinic
2
o
stirred at 40 C for 3 days and dialyzed for 3 days (MWCO, dehydrogenase enzymes within the viable cells, precipitating
7
kDa). The PEG-b-PBLG polymer was obtained by freeze the insoluble formazan crystals. After removal of the minimal
drying, and the degree of polymerization of PBLG was 50, essential medium, an ethanol–dimethyl sulfoxide solution
1
and characterized by H NMR, gel permeation chromatog- (1:1) was added to each well. After complete solubilization
raphy (GPC) and Fourier transform infrared spectroscopy of the dark blue crystals of MTT formazan (20 minutes),
(
(
FT-IR).
the absorbance of the content of each well was measured
The proper amounts of PEG-b-PBLG in DMF at 570 nm on a multimode reader. The blank reference was
0.1 g/3.5 mL) were mixed with five times the amount of taken from wells without cells, which were also incubated
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International Journal of Nanomedicine 2016:11
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Tonꢇ et al
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with the MTT solution. Polymers were used as a positive
cytotoxicity control. Cells cultivated on the wells were used
as a negative cytotoxicity control. Values were expressed as
optical density (OD). Data were subtracted from the blank
ethanol-dimethyl sulfoxide absorbance, and presented as a
percentage relative to control as is seen in equation 1:
room temperature with 6 mL of FITC-labelled insulin-loaded
polymer against 80 mL of 0.02 mol/L phosphate buffer (pH
7
.4). At 0.5, 1, 2, 3, 6, 12, 24, 48, 72, 96, and 120 hour time
intervals, a given volume of release medium was withdrawn
and replenished with an equal volume of fresh release
medium. The amounts of released FITC-labelled insulin and
FITC-labelled insulin remaining in the dialysis tube were
(1) determined by an ultraviolet fluorescence scanning enzyme
standard instrument. The release of free FITC-labelled insulin
in the absence of polymer was also performed as a control.
All of the experiments were conducted in duplicate.
ODsample − ODblank
(
%) Cell Viability =
×100
ODcells − ODblank
ꢃncapsulation of insulin by Pꢃg-b-
Pꢃꢂg-ꢇ-Pꢂꢂ)
(
To evaluate the encapsulation of insulin by PEG-b-(PELG-g- References
1
. Hideharu M, Mizuki I, Satoko I, Takeshi E. Ring-opening polymerization
PLL), a given volume of fluorescein isothiocyanate (FITC)-
labelled insulin (2 mg/mL) in phosphate buffer at 0.01
mmol/L (pH 7.2) was added to PEG-b-(PELG-g-PLL) in a
phosphate buffer. After 30 minutes, the mixed solution was
transferred to dialysis tubing (MWCO, 100 kDa) and dialyzed
against a 0.1 mol/L phosphate buffer under sink conditions.
The dialysis of free FITC-labelled insulin in phosphate buffer
was also conducted under the same conditions as the control.
When the free FITC-labelled insulin in the control experiment
were completely removed, the polymer-encapsulated FITC-
labelled insulin was determined by an ultraviolet fluorescence
scanning enzyme standard instrument, and characterized by
a nanoparticle size analyzer, transmission electron micro-
scopy and FT-IR. All of the experiments were carried out
in duplicate.
of γ-benzyl-l-glutamate-N-carboxyanhydride in ionic liquids. Polymer.
2007;48(20):5867–5877.
. Poché DS, Moore MJ, Bowles JL. An unconventional method for purify-
ing the Ncarboxyanhydride derivatives of γ-alkyl-L-glutamates. Synth
Commun. 1999;29(5):843–854.
3. Zhuang W, Liao L, Chen H, Wang J, Pan Y, Zhang L, et al. Water-soluble
star-block copolypeptides: towards biodegradable nanocarriers for ver-
satile and simultaneous encapsulation. Macromol Rapid Commun. 2009;
30:920–924.
. Harada A, Kataoka K. Formation of polyion complex micelles in an
aqueous milieu from a pair of oppositely-charged block copolymers
with poly(ethylene glycol) segments. Macromolecules. 1995;28(15):
5294–5299.
2
4
5
6
. Wu M, Ye Z, Liu Y, Liu B, Zhao X. Release of hydrophobic anticancer
drug from a newly designed self-assembling peptide. Mol Biosyst.
2011;7(6):2040–2047.
. Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J Immunol Methods.
1
983;65(1–2):55–63.
In vitro release of insulin from Pꢃg-b-
(
Pꢃꢂg-g-Pꢂꢂ)
The release of insulin from the PEG-b-(PELG-g-PLL) was
investigated using a dialysis method (MWCO, 100 kDa) at
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