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

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: HIF-1α; RI/RILI; VEGF; block copolymer; insulin.

Figure 1

(A) Encapsulation of insulin by linear-brush block copolymers. (B) The structure of PEG-b-(PELG-g-PLL). Note: m and n represent the degree of polymerization. Abbreviation: PEG-b-(PELG-g-PLL), poly(ethylene glycol)-b-(poly(ethy lenediamine l-glutamate)-g-poly(l-lysine)).

Figure 2

Synthesis of linear-brush copolymer of PEG-b-(PELG-g-PLL). Abbreviations: BLG, γ-benzyl l-glutamate; BTC, bis(trichloromethyl)carbonate; BLG-NCA, γ-benzyl l-glutamate-N-carboxyanhydride; PEG-NH₂: poly(ethylene glycol) amine; PEG-b-PBLG, poly(ethylene glycol)-b-poly(γ-benzyl l-glutamate); DMF, N, N-dimethylformamide; 2-HP, 2-hydroxypyridine; EA, ethylenediamine; PEG-b-PELG, poly(ethylene glycol)-b-poly(ethylenediamine l-glutamate); ZLL-NCA, ε-benzyoxycarbonyl-l-lysine-N-carboxyanhydride; PEG-b-(PELG-g-PZLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)); PEG-b-(PELG-g-PLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)).

Figure 3

¹H NMR spectra of block copolymer PEG-b-(PELG-g-PLL) and its intermediates. Notes: (A) PEG-b-PBLG in CDCl₃; (B) PEG-b-PELG in D₂O; (C) PEG-b-(PELG-g-PZLL) in DMSO-d₆; (D) PEG-b-(PELG-g-PLL) in D₂O. Peak labels (a–k) in each spectrum correspond to the labels (a–k) in the inserted structural formula. Abbreviations: ¹H NMR, ¹H-nuclear magnetic resonance; PEG-b-PBLG, poly(ethylene glycol)-b-poly(γ-benzyl l-glutamate); PEG-b-PELG, poly(ethylene glycol)-b-poly(ethylenediamine l-glutamate); PEG-b-(PELG-g-PZLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)); PEG-b-(PELG-g-PLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)); DMSO, dimethylsulfoxide.

Figure 4

GPCs of (A) PEG-b-PBLG; (B) PEG-b-PELG; (C) PEG-b-(PELG-g-PZLL); and (D) PEG-b-(PELG-g-PLL). Abbreviations: GPC, gel permeation chromatogram; PEG-b-PBLG, poly(ethylene glycol)-b-poly(γ-benzyl l-glutamate); PEG-b-PELG, poly(ethylene glycol)-b-poly(ethylenediamine l-glutamate); PEG-b-(PELG-g-PZLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(ε-benzyoxycarbonyl-l-lysine)); PEG-b-(PELG-g-PLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)).

Figure 5

Characterization of PEG-b-(PELG-g-PLL) and insulin/PEG-b-(PELG-g-PLL) complexes. Notes: (A) Cellular viability of EC9706 cells cultured with different concentrations of PEG-b-(PELG-g-PLL); (B) TEM image of insulin/PEG-b-(PELG-g-PLL) complexes; (C) Diameter of block copolymer PEG-b-(PELG-g-PLL) in PB; (D) Diameter of insulin/PEG-b-(PELG-g-PLL) complexes in PB; (E) Cumulative releasing profile of insulin from insulin/PEG-b-(PELG-g-PLL) complexes. Abbreviations: TEM, transmission electron microscopy; PB, phosphate buffer; PEG-b-(PELG-g-PLL), poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)).

Figure 6

Levels of serum UN (A) and Scr (B). Notes: The serum of Sham, I/R, Insulin, and Insulin/polymer groups rats was collected 24 hours after reperfusion and results expressed as mean ± SD. (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/R groups is denoted by **P<0.01, and a significant decrease from I/R groups is denoted by ***P<0.01. Abbreviations: BUN, blood urea nitrogen; Scr, serum creatinine; I/R, ischemia/reperfusion; SD, standard deviation.

Figure 7

The level of D/W lung ratios for different groups of rats. Notes: The pulmonary tissues of Sham, I/R, Insulin, and Insulin/polymer groups rats were collected 24 hours after reperfusion and the levels of D/W lung ratios measured. Results are expressed as mean ± SD. A significant decrease from Sham group 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 ***P<0.01. Abbreviations: D/W, dry/wet; I/R, ischemia/reperfusion; SD, standard deviation.

Figure 8

The activity of SOD and the level of MDA in pulmonary tissues for different groups of rats. Notes: The pulmonary tissues of Sham, I/R, Insulin, and Insulin/polymer groups rats were collected 24 hours after reperfusion and the activity of SOD and the level of MDA measured. Results are expressed as mean ± SD. (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: SOD, superoxide dismutase; I/R, ischemia/reperfusion; MDA, methane dicarboxylic aldehyde; SD, standard deviation.

Figure 9

The level of TNF-α and the activity of MPO in pulmonary tissues for different groups of rats. Notes: The pulmonary tissues of Sham, I/R, Insulin and Insulin/polymer groups rats were collected 24 hours after reperfusion and the level of TNF-α and the activity of MPO measured. Results are expressed as mean ± SD. (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/R groups 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/R, ischemia/reperfusion; MPO, myeloperoxidase; SD, standard deviation.

Figure 10

The qualitative expression (C) and quantitative analyses of HIF-1α (A) and VEGF (B) in pulmonary tissues for different groups of rats. Notes: The pulmonary tissues of Sham, I/R, Insulin, and Insulin/polymer groups rats were collected 24 hours after reperfusion and the expression of HIF-1α and VEGF measured. Results are expressed as mean ± SD. (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/R groups is denoted by **P<0.01, and a significant decrease from I/R groups is denoted by ***P<0.01. Abbreviations: HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor; I/R, ischemia/reperfusion; SD, standard deviation.