Yue Tong

Chitosan-coated liposomes: characterization and interaction with leuprolide

The objective of the present work was to investigate the effect of chitosan concentration and lipid type on the characteristics of chitosan-coated liposomes and their interactions with leuprolide. Liposomes from lipid of high purity and low purity were prepared and coated by chitosan. Physical properties, drug entrapment efficiency, and stability upon dilution were respectively compared. Results showed that the particle size increment of liposomes from low purity lipid was larger than that from high purity lipid, indicating a thicker coating layer. The high zeta potential of particles from low purity lipid was thought to play an important role in the resistance to flocculation. As to particles from high purity lipid, polymer bridging caused flocculation at low polymer concentration while at high concentration, the adsorbed chitosan molecule led to steric stabilization. Drug entrapment efficiency decreased as chitosan was added to liposomes, showing the disturbance of bilayers. Upon dilution, the leakage of leuprolide from low purity liposomes was larger than that from high purity liposomes. In conclusion, low purity lipid possessed more negative charge and formed thicker adsorptive layer by stronger electrostatic attraction with chitosan. The interaction between chitosan and the polar head groups on the surface of phospholipid bilayers may interfere with leuprolide entrapped in liposomes and result in the leakage of leuprolide.

Characterization of a monoPEG20000-Endostar

In this study, we investigated the PEG attachment site of mono-PEGylated Endostar, a modified recombinant human endostatin approved in China for lung cancer. N-terminal site-directed mono-PEGylation of Endostar was accomplished using mPEG-propionaldehyde derivatives (Mw=20 kDa) under slightly acidic pH conditions (pH 5.5). One-step cation exchange chromatography was used to purify the mono-PEGylated Endostar. Following tryptic digestion, the peptide fragment containing PEG was separated by SDS-PAGE. Barium iodide staining and Western blotting were used to detect the PEG moiety and the N-terminus of Endostar, respectively. The peptide fragment stained by barium iodide showed a positive response to anti-(His) 6 mAb, demonstrating that PEG was located at the N-terminus of Endostar. LC/MS was applied to verify the occurrence of mono-PEGylation at the N-terminus of Endostar.

Characterization of a site-specific PEGylated analog of exendin-4 and determination of the PEGylation site.

PEGylation has been a successful strategy for improving the pharmacokinetic and pharmaceutical properties of proteins and peptides. However, PEGylated products also create significant challenges for detailed structural characterization. In this work, a site-specific PEGylation strategy was successfully performed on an exendin-4 analog (Ex4C) through a maleimide method. Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), analytical reversed phase HPLC (RP-HPLC) and MALDI-TOF were applied to verify the accomplishment of the PEGylation. Peptide mapping was investigated after tryptic digestion, and the PEGylaton site was successfully located on the C-terminal fragment of Ex4C. Amino acid analysis (AAA) of cysteine was then applied to verify the block in the thiol group caused by PEGylation. We believe that the combination of proper enzymatic digestion and amino acid analysis of cysteine provided an easy and convincing way to identify the PEGylation site in this maleimide method.

A site-specific PEGylated analog of exendin-4 with improved pharmacokinetics and pharmacodynamics in vivo

Objectives  Our aim was to improve the in vivo pharmacokinetics and pharmacodynamics of exendin‐4 by using site‐specific PEGylation.

Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein

Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.

Improving the anti-diabetic activity of GLP-1 by fusion with globular adiponectin.

Abstract Glucagon-like peptide-1 (GLP-1) as an endogenous glucose-lowering peptide has great potential in diabetes therapy, but its clinical utility is compromised by its limited activity in vivo. Herein to improve the anti-diabetic activity of GLP-1, we constructed a fusion protein (GLP-1-globular adiponectin, GAD) using this peptide and the globular adiponectin. Using recombinant expression, we prepared the GAD fusion protein with a purity of above 95%. In normal mice, we validated the acute glucose-lowering activity of GAD by performing intraperitoneal glucose tolerance test (IPGTT). After that, we evaluated the anti-diabetic activity of this fusion protein in a multiple-low-dose streptozotocin (STZ)-induced diabetic mice model. In this diabetic mice model, GAD treatment greatly reduced the elevated fasting glucose and improved their impaired glucose homeostasis as judged by oral glucose tolerance test (OGTT). After treatment, the fasting glucose was 15.34 ± 2.07 mmol/L and 9.47 ± 1.08 mmol/L for GLP-1-treated and GAD-treated diabetic mice, respectively. Moreover, GLP-1 treatment and GAD treatment improved the pancreas function of the diabetic mice by ~1.5-fold and ~4.2-fold, respectively. Furthermore, GAD treatment greatly reduced the tissue damage in the pancreas of the diabetic mice. These data suggest that GAD possesses an improved anti-diabetic activity in vivo compared with native GLP-1, and therefore it could be regarded as a potential candidate for the future development of anti-diabetic drugs.

Alleviation of high-fat diet-induced atherosclerosis and glucose intolerance by a novel GLP-1 fusion protein in ApoE −/− mice

We have previously constructed an engineered anti-diabetic fusion protein using glucagon-like peptide-1 and the globular domain of adiponectin. Herein, we evaluated the therapeutic effects of this fusion protein (GAD) on high-fat diet (HFD)-fed ApoE−/− mice. The lipid-lowering effect of GAD was determined in C57BL/6 mice using a lipid tolerance test. The effects of GAD on HFD-induced glucose intolerance, atherosclerosis, and hepatic steatosis were evaluated in HFD-fed ApoE−/− mice using glucose tolerance test, histological examinations and real-time quantitative PCR. The anti-inflammation activity of GAD was assessed in vitro on macrophages. GAD improved lipid metabolism in C57BL/6 mice. GAD treatment alleviated glucose intolerance, reduced blood lipid level, and attenuated atherosclerotic lesion in HFD-fed ApoE−/− mice, which was associated with a repressed macrophage infiltration in the vessel wall. GAD treatment also blocked hepatic macrophage infiltration and prevented hepatic inflammation. GAD suppressed lipopolysaccharide-triggered inflammation responses on macrophages, which can be abolished by H89, an inhibitor of protein kinase A. These findings demonstrate that GAD is able to generate a variety of metabolic benefits in HFD-fed ApoE−/− mice and indicate that this engineered fusion protein is a promising lead structure for anti-atherosclerosis drug discovery.

PEGylation-aided refolding of globular adiponectin

Globular adiponectin (GAD) as the active domain of adiponectin is a promising candidate for anti-diabetic drug development. The recombinant production of GAD in Escherichia coli, however, is difficult because it is mainly expressed as inclusion bodies which need to be refolded to regain function. In this study we developed a novel method for refolding of GAD with a high efficiency by using polyethylene glycol (PEG) conjugation. An artificially designed DNA sequence encoding for GAD was synthesized and inserted into the pET28a vector to construct an expression plasmid which was thereafter transformed into E. coli BL21 (DE3) host cells for heterologous expression. After bacterial cell culture employing auto-induction medium, the inclusion bodies were collected, washed and dissolved in guanidine hydrochloride before PEG conjugation. Then the PEG-conjugated GAD was refolded by dialysis and purified by two steps of chromatography. The refolded conjugate showed a marked glucose-lowering activity in mice, demonstrating that it had been successfully refolded. As a convenient method, PEGylation-aided refolding could also be tested on other proteins to explore its suitability.

A mutated glucagon‐like peptide‐1 with improved glucose‐lowering activity in diabetic mice

The aim of this study was to characterize the conformation and potency of a mutated glucagon‐like peptide‐1 (mGLP‐1), and evaluate its glucose‐lowering activity in diabetic mice.

Development of a C-Terminal Site-Specific PEGylated Analog of GLP-1 with Improved Anti-Diabetic Effects in Diabetic Mice

Preclinical Research