Jinhong Zheng

A poly(L-lysine)-based hydrophilic star block co-polymer as a protein nanocarrier with facile encapsulation and pH-responsive release.

A hydrophilic star block co-polymer was synthesized, characterized, and evaluated as a protein nanocarrier. The star block co-polymer was composed of a hyperbranched polyethylenimine (PEI) core, a poly(L-lysine) (PLL) inner shell, and a poly(ethylene glycol) (PEG) outer shell. The model protein insulin can be rapidly and efficiently encapsulated by the synthesized polymer in aqueous phosphate buffer at physiological pH. Complexation between PEI-PLL-b-PEG and insulin was investigated using native polyacrylamide gel electrophoresis. The uptake of enhanced green fluorescent protein into Ad293 cells mediated by PEI-PLL-b-PEG was also investigated. The encapsulated insulin demonstrated sustained release at physiological pH and showed accelerated release when the pH was decreased. The insulin released from the star block co-polymer retained its chemical integrity and immunogenicity.

The protective effects of N-n-butyl haloperidol iodide on myocardial ischemia-reperfusion injury in rats by inhibiting Egr-1 overexpression.

Aims: Our previous studies have shown that N-n-butyl haloperidol iodide (F2) can antagonize myocardial ischemia/reperfusion (I/R) injury by blocking intracellular Ca2+ overload. The present study is to test the hypothesis that the protective effects of F2 on myocardial I/R injury is mediated by downregulating Egr-1 expression. Methods: The Sprague–Dawley rat myocardial I/R model and cardiomyocyte hypoxia/reoxygenation (H/R) model were established. With antisense Egr-1 oligodeoxyribonucleotide (ODN), the relationship between Egr-1 expression and myocardial I/R injury was investigated. Hemodynamic parameters, myeloperoxidase (MPO), cardiac troponin I (cTnI) and tumor necrosis factor-α (TNF-α) were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Egr-1 mRNA and protein expressions were examined by Northern-blot and Western-blot analyses. Results: Treatment with antisense Egr-1 ODN significantly reduced Egr-1 protein expression and attenuated injury of myocardial tissues and cells. Meanwhile, treatment with F2 significantly inhibited the overexpression of Egr-1 mRNA and protein in myocardial tissues and cells. Consistent with downregulation of Egr-1 expression by F2, inflammation and other damages were significantly relieved evidenced by the amelioration of hemodynamics, the reduction in myocardial MPO activity as well as the decrease in leakage of cTnI and release of TNF-α from cardiomyocyte. Conclusions: These results suggested that the overexpression of Egr-1 was causative in myocardial I/R or H/R injury, and F2 could protect myocardial tissues and cells from I/R or H/R injury, which was largely due to the inhibition of Egr-1 overexpresssion.

Poly(L-lysine)-based star-block copolymers as pH-responsive nanocarriers for anionic drugs.

Star-block copolymers PEI-g-(PLL-b-PEG) with a branched polyethylenimine (PEI) core, a poly(l-lysine) (PLL) inner shell, and a poly(ethylene glycol) (PEG) outer shell have been synthesised and evaluated as potential nanocarriers for anionic drugs. The star-block copolymers were synthesised by a ring-opening polymerisation of ɛ-benzyloxycarbonyl-L-lysine N-carboxyanhydride initiated by the peripheral primary amino groups of PEI, surface modification with activated PEG 4-nitrophenyl carbonate, and subsequent deprotection of benzyl groups on the side chains of the PLL inner shell. The synthesised star-block copolymers were characterised by (1)H NMR, gel permeation chromatography (GPC), and dynamic light scattering (DLS). The encapsulation properties of these star-block copolymers were characterised by spectrophotometric titration and dialysis. These techniques demonstrated that anionic model dyes, such as methyl orange and rose Bengal, and the model drug diclofenac sodium can be encapsulated efficiently by PEI-g-(PLL-b-PEG) at physiological pH. The entrapped model compounds demonstrated sustained release at physiological pH and accelerated release when the pH was either increased to 10.0-11.0 or decreased to 2.0-3.0. The efficient encapsulation as well as the pH-responsive releasing properties of these star-block copolymers could be potentially used in the controlled release of anionic drugs.

The Protective Effect of Egr-1 Antisense Oligodeoxyribonucleotide on Myocardial Injury Induced by Ischemia-reperfusion and Hypoxia- reoxygenation

Aims: Our previous studies have shown that myocardial ischemia-reperfusion (I/R) injury is related closely with early growth response (Egr)-1 overexpression. The present study is to confirm thoroughly the effects of Egr-1 on the occurrance and development of myocardial I/R injury. Methods: The Sprague-Dawley rat myocardial I/R model and cultured cardiomyocyte hypoxia-reoxygenation (H/R) model were established. The synthesized Egr-1 antisense oligodeoxyribonucleotide (AS-ODN) was transfected into myocardial tissues and cells. Hemodynamic parameters, myeloperoxidase (MPO), cardiac troponin I (cTnI), tumor necrosis factor-α (TNF-α), morphology, spontaneous beat and cell viability were measured to assess the degree of injury and inflammation of myocardial tissues and cells. Results: In vivo, Egr-1 AS-ODN significantly attenuated injury and inflammation of myocardial tissues caused by I/R evidenced by the amelioration of hemodynamics and the reduction in MPO activity. In vitro, Egr-1 AS-ODN significantly relieved injury and inflammation of cultured cardiomyocyte caused by H/R evidenced by the improvement in morphology, structure and beat as well as the decrease in leakage of cTnI and release of TNF-α from cultured cardiomyocyte. Conclusions: These data suggest that Egr-1 plays a vital role in the pathogenesis of myocardial I/R injury and Egr-1 AS-ODN could protect the myocardium from I/R injury.

N-4-tert-butyl benzyl haloperidol chloride suppresses Ca2+-dependent Egr-1 expression and subsequently inhibits vascular smooth muscle cell proliferation induced by angiotensin II.

Background: N-4-Tert-Butyl benzyl haloperidol chloride (C3) was a novel calcium antagonist synthesized in our laboratory. The present study is to explore the effect of C3 on vascular smooth muscle cell proliferation and the mechanism involved. Methods: The effects of C3 on Ang II-induced cytosolic free Ca2+ concentration change, VSMC proliferation, the key early growth response factor 1 (Egr-1) were evaluated by laser scanning confocal microscopy, microtiter tetrazolium (MTT) proliferation assay, flow cytometry analysis, Western blot and RT-PCR analysis, respectively. An extracellular Ca2+ chelator EGTA and antisense Egr-1 oligodeoxyribonucleotides (ODNs) were used to establish the relation between Ca2+-dependent Egr-1 expression induced by Ang II and VSMC proliferation. Results: C3 attenuated the Ang II-induced extracellular Ca2+ influx, inhibited VSMCs proliferation and arrested VSMCs in G1-phase. C3 also triggered a significant reduction in PDGF-A and cyclin D1, Cdk2 along with an overexpression of p21Cip1. Antisense Egr-1 ODNs inhibited VSMCs proliferation, which was related to G1-phase arrest, due to inhibiting the expression of Egr-1 and C3 inhibited the overexpression of Egr-1. Conclusion: Egr-1 may play a key role in Ang II-induced proliferation of VSMCs. C3 inhibits vascular smooth muscle cell proliferation and the mechanism is involved with the inhibition of over-expression of Egr-1.

N-n-butyl Haloperidol Iodide Protects Cardiac Microvascular Endothelial Cells From Hypoxia/Reoxygenation Injury by Down-Regulating Egr-1 Expression

Aims: Our previous studies have shown that N-n-butyl haloperidol iodide (F2) can antagonize myocardial ischemia/reperfusion (I/R) injury by down-regulating the early growth response (Egr)-1 expression, but the molecular mechanisms are not well understood. Because there is evidence implicating myocardial I/R injury is closely associated with endothelial dysfunction. The present study is to test the hypothesis that the protective effects of F2 on myocardial I/R injury is related closely with down-regulating Egr-1 expression on cardiac microvascular endothelial cells (CMECs). Methods: A model of cultured CMECs exposed to hypoxia/reoxygenation (H/R) was developed. With antisense Egr-1 oligodeoxyribonucleotide (ODN), the relationship between Egr-1 expression and endothelial H/R injury was investigated. Egr-1 mRNA and protein expression were examined by real-time fluorescent quantitative PCR, immunocytochemical staining and Western-blot analysis. Lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), intercellular adhesion molecule-1 (ICAM-1), adherence of neutrophil and platelets, and cell viability were measured after H/R to evaluate the degree of endothelial injury. Results: Pretreatment with antisense Egr-1 ODN significantly reduced Egr-1 protein expression and attenuated injury of CMECs. Consistent with down-regulation of Egr-1 expression by F2, inflammation and other damage were significantly reduced as evidenced by a decrease of ICAM-1 expression, reduction of neutrophil and platelets adherence, increase in SOD, and decreases in MDA and LDH levels, resulting in the rise of cell viability. Conclusions: We demonstrate a protective effect of F2 in CMECs against H/R injury by down-regulating Egr-1 expression, which might be play a vital role in the pathogenesis of myocardial I/R injury.

Controlled biosilification using self-assembled short peptides A6K and V6K

We report the molecular self-assembly of two amphiphilic peptides (A6K and V6K) and the application of their self-assemblies as organic templates to direct biosilica formation. Under ambient conditions, A6K self-assembled into nanotubes 2.7 nm tall and approximately 1 μm to 2 μm long. In contrast, V6K self-assembled into lamellar-stack nanostructures approximately 4 nm tall and under 100 nm long. The self-assembled peptide nanostructures were used as organic templates to direct biosilica formation. Comparing with the self-assembled structures formed by the peptide/anions system, novel silica morphologies can be obtained by changing the peptide composition, using different anions, and applying electrostatic/flow fields. We observed that the presence of anions is important but not enough to produce ordered silica structures with novel morphologies. This study provides further understanding of silica biomineralization tailored by assembled peptides, which offers a simple but efficient method to control the formation of inorganic material.

N-n-Butyl haloperidol iodide protects against hypoxia/reoxygenation-induced cardiomyocyte injury by modulating protein kinase C activity

N-n-Butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. We tested whether the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by modulating protein kinase C (PKC) activity in primary cultured cardiomyocytes. Primary cultures of ventricular cardiomyocytes underwent 2-h hypoxia and 30-min reoxygenation. Total PKC activity was measured, and the translocation pattern of PKCalpha, betaII, delta and epsilon isoforms was assessed by fractionated western blot analysis. We investigated the association of PKC isoform translocation and H/R-induced injury in the presence and absence of the specific inhibitors and activator. Measurements included cell damage evaluated by creatine kinase (CK) release, and apoptosis measured by annexin V-FITC assay. In primary cultured cardiomyocytes exposed to H/R, PKCalpha, delta and epsilon were translocated, with no change in PKCbetaII activity. Total PKC activity, CK release and apoptosis were increased after H/R. Treatment with the conventional PKC inhibitor Go6976 reduced early growth response-1 (Egr-1) protein expression and attenuated apoptosis. The PKCepsilon inhibitor peptide epsilonV1-2 increased H/R injury without influencing Egr-1 expression. Pretreatment with F2 inhibited translocation of PKCalpha, increased translocation of PKCepsilon, and relieved the CK release and apoptosis. The protection of F2 was blocked in part by the conventional PKC activator thymeleatoxin (TXA) and epsilonV1-2 peptide. F2 significantly alleviated H/R-induced injury, which might be attributed to the combined benefits of inhibiting PKCalpha and activating PKCepsilon.

Synthesis and Evaluation of New Pyrazoline Derivatives as Potential Anticancer Agents in HepG-2 Cell Line

Cancer is a major public health concern worldwide. Adverse effects of cancer treatments still compromise patients’ quality of life. To identify new potential anticancer agents, a series of novel pyrazoline derivatives were synthesized and evaluated for cytotoxic effects on HepG-2 (human liver hepatocellular carcinoma cell line) and primary hepatocytes. Compound structures were confirmed by 1H-NMR, mass spectrometry, and infrared imaging. An in vitro assay demonstrated that several compounds exerted cytotoxicity in the micromolar range. Benzo[b]thiophen-2-yl-[5-(4-hydroxy-3,5-dimethoxy-phenyl)-3-(2-hydroxy-phenyl)-4,5-dihydo-pyrazol-1-yl]-methanone (b17) was the most effective anticancer agent against HepG-2 cells owing to its notable inhibitory effect on HepG-2 with an IC50 value of 3.57 µM when compared with cisplatin (IC50 = 8.45 µM) and low cytotoxicity against primary hepatocytes. Cell cycle analysis and apoptosis/necrosis evaluation using this compound revealed that b17 notably arrested HepG-2 cells in the G2/M phase and induced HepG-2 cells apoptosis. Our findings indicate that compound b17 may be a promising anticancer drug candidate.

The Synthesis of a Novel Chalcone and Evaluation for Anti-free Radical Activity and Antagonizing the Learning Impairments in Alzheimer's Model

We synthesized a new chalcone (4,2’-dihydroxy-3methoxy-5-bromine chalcone; C) and structurally identified it via infrared spectrometry (IR), 1H-NMR, mass spectrometry (MS) and element analysis (EA). C was confirmed to be highly potent in scavenging 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and OH free radicals in vitro. Tests of anti-free radical activity in response to oxidative stress in mice revealed that C could elevate glutathione peroxidase (GSH-PX) and super oxide dismutase (SOD) levels and lower malonaldehyde (MDA) level in a free-radical–injured scopolamine-induced Alzheimer’s model. Further behavioral tests with the Morris water maze showed that C could antagonize the learning impairments in the Alzheimer’s model, which suggests that C has a potential role in Alzheimer’s disease.