Jing Sheng

Biscarbamate cross-linked polyethylenimine derivative with low molecular weight, low cytotoxicity, and high efficiency for gene delivery

Polyethylenimine (PEI), especially PEI 25 kDa, has been widely studied for delivery of nucleic acid drugs both in vitro and in vivo. However, it lacks degradable linkages and is too toxic for therapeutic applications. Hence, low-molecular-weight PEI has been explored as an alternative to PEI 25 kDa. To reduce cytotoxicity and increase transfection efficiency, we designed and synthesized a novel small-molecular-weight PEI derivative (PEI-Et, Mn: 1220, Mw: 2895) with ethylene biscarbamate linkages. PEI-Et carried the ability to condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed complete condensation of pDNA at w/w ratios that exceeded three. The particle size of polymer/pDNA complexes was between 130 nm and 180 nm and zeta potential was 5–10 mV, which were appropriate for cell endocytosis. The morphology of PEI-Et/pDNA complexes observed by atomic force microscopy (AFM) was spherically shaped with diameters of 110–190 nm. The transfection efficiency of polymer/pDNA complexes as determined with the luciferase activity assay as well as fluorescence-activated cell-sorting analysis (FACS) was higher than commercially available PEI 25 kDa and Lipofectamine 2000 in various cell lines. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration in three cell lines. Therefore, our results indicated that the PEI-Et would be a promising candidate for safe and efficient gene delivery in gene therapy.

Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)- graft-polyethylenimine derivative

Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79–100 nm and zeta potential was 6–15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53–65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery.

Delivery of Therapeutic AGT shRNA by PEG-Bu for Hypertension Therapy

Gene silencing by RNA interference (RNAi) is a promising approach for gene therapy. However, up to today, it is still a major challenge to find safe and efficient non-viral vectors. Previously, we reported PEI-Bu, a small molecular weight PEI derivative, as an efficient non-viral carrier. However, like many PEI-based polymers, PEI-Bu was too toxic. In order to reduce cytotoxicity while maintain or even enhance transfecion efficiency, we modified PEI-Bu with poly(ethylene glycol) (PEG) to obtain PEG-Bu, and used it to delivery a theraputic short hairpin RNA (shRNA) targeting angiotensinogen (AGT) to normal rat liver cells (BRL-3A), which was a key target for the treatment of hypertension. The structure of PEG-Bu was confirmed by proton nuclear magnetic resonance (1H-NMR). Gel permeation chromatography (GPC) showed that the weight average molecular weight (Mw) of PEG-Bu was 5880 Da, with a polydispersity of 1.58. PEG-Bu could condense gene cargo into spherical and uniform nanoparticles with particle size (65–88 nm) and zeta potential (7.3–9.6 mV). Interestingly and importantly, PEG-Bu displayed lower cytotoxicity and enhanced tranfection efficiency than PEI-Bu after PEGylation in both normal cells BRL-3A and tumor cells HeLa. Moreover, PEG-Bu could efficiently delivery AGT shRNA to knockdown the AGT expression. To sum up, PEG-Bu would be a promising non-viral vector for delivering AGT shRNA to BRL-3A cells for hypertension therapy.

Targeting the hedgehog signaling pathway for cardiac repair and regeneration

The hedgehog (Hh) signaling pathway is involved in the angiogenesis and development of the coronary vasculature in the embryonic heart. Recently, the Hh signal pathway has emerged as an important regulator that can increase cardiomyocyte proliferation, inhibit cardiomyocyte death and apoptosis, recruit endothelial progenitor cell (EPCs) into sites of myocardial ischemia, and direct stem cells to differentiate into cardiac muscle lineage. Experimental studies have tried to target the Hh signaling pathway for cardiac repair and regeneration. The purpose of this review is to discuss the role of the Hh signal pathway in cardiac repair and regeneration as well as the current strategies targeting the Hh signaling pathway and its potential in heart diseases.ZusammenfassungDer Hedgehog(Hh)-Signalweg ist an der Angiogenese und Entwicklung des koronaren Gefäßsystems im embryonalen Herzen beteiligt. Vor Kurzem hat sich der Hh-Signalweg als ein bedeutender Regulator erweisen, der die Kardiomyozytenproliferation erhöhen, Tod und Apoptose von Kardiomyozyten hemmen, endotheliale Vorläuferzellen („endothelial progenitor cells“, EPC) an Orte myokardialer Ischämie locken und Stammzellen so steuern kann, dass sie zu Zellen der Herzmuskelzellreihe ausdifferenzieren. In experimentellen Studien wurde versucht, auf den Hh-Signalweg bei der kardialen Wiederherstellung und Regeneration abzuzielen. Ziel der vorliegenden Übersichtsarbeit ist es, die Bedeutung des Hh-Signalwegs bei der kardialen Wiederherstellung und Regeneration sowie aktuelle Ansätze, die auf den Hh-Signalweg und sein Potenzial bei Herzerkrankungen abzielen, zu erörtern.

SIRT1 inhibits TGF-β-induced endothelial-mesenchymal transition in human endothelial cells with Smad4 deacetylation: LI et al.

Endothelial‐mesenchymal transition (EndMT) plays a pivotal role in organ fibrosis. This study examined the effect of SIRT1 on transforming growth factor beta (TGF‐β)‐induced EndMT in human endothelial cells (ECs) and its probable molecular mechanism. We assessed EndMT by immunofluorescence staining, quantitative real‐time polymerase chain reaction, Western blotting, and migration and invasion assays. Adenovirus was used to overexpress or knockdown SIRT1 in ECs. The regulatory relationship between SIRT1 and Smad4 was analyzed by coimmunoprecipitation assay. We found that SIRT1 was decreased in TGF‐β‐induced EndMT, and SIRT1 inhibited TGF‐β‐induced EndMT through deacetylating Smad4. Our findings suggest that SIRT1 has an important role in inhibiting EndMT by regulating the TGF‐β/Smad4 pathway in human ECs and, thus, protecting against fibrosis.

Effects of the visual-feedback-based force platform training with functional electric stimulation on the balance and prevention of falls in older adults: a randomized controlled trial

Background Force platform training with functional electric stimulation aimed at improving balance may be effective in fall prevention for older adults. Aim of the study is to evaluate the effects of the visual-feedback-based force platform balance training with functional electric stimulation on balance and fall prevention in older adults. Methods A single-centre, unblinded, randomized controlled trial was conducted. One hundred and twenty older adults were randomly allocated to two groups: the control group (n = 60, one-leg standing balance exercise, 12 min/d) or the intervention group (n = 60, force platform training with functional electric stimulation, 12 min/d). The training was provided 15 days a month for 3 months by physical therapists. Medial–lateral and anterior–posterior maximal range of sway with eyes open and closed, the Berg Balance Scale, the Barthel Index, the Falls Efficacy scale-International were assessed at baseline and after the 3-month intervention. A fall diary was kept by each participant during the 6-month follow-up. Results On comparing the two groups, the intervention group showed significantly decreased (p < 0.01) medial–lateral and anterior–posterior maximal range of sway with eyes open and closed. There was significantly higher improvement in the Berg Balance Scale (p < 0.05), the Barthel Index (p < 0.05) and the Falls Efficacy Scale-International (p < 0.05), along with significantly lesser number of injurious fallers (p < 0.05), number of fallers (p < 0.05), and fall rates (p < 0.05) during the 6-month follow-up in the intervention group. Conclusion This study showed that the visual feedback-based force platform training with functional electric stimulation improved balance and prevented falls in older adults.

Local honokiol application inhibits intimal thickening in rabbits following carotid artery balloon injury

Honokiol is a natural bioactive product with anti-tumor, anti-inflammatory, anti-oxidative, anti-angiogenic and neuroprotective properties. The present study aimed to investigate the effects of honokiol treatment on intimal thickening following vascular balloon injury. The current study determined that perivascular honokiol application reduced intimal thickening in rabbits 14 days after carotid artery injury, it may inhibit vascular smooth muscle cell (VSMCs) proliferation and reduce collagen deposition in local arteries. The findings of the presents study also suggested that honikiol may increase the mRNA expression levels of matrix metalloproteinase-1 (MMP-1), MMP-2 and MMP-9 and decrease tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression in the rabbit arteries. Additionally, perivascular honokiol application inhibited intimal thickening, possibly via inhibition of the phosphorylation of SMAD family member 2/3.

Screening PEGylated polyethylenimine derivatives for safe and efficient delivery of gene materials

Gene-based therapy has broad and important clinical applications, and the non-viral vector delivery of exogenous nucleic acids is commonly used for gene therapy. Among the numerous gene delivery vectors, the cationic polymer vectors are considered to be the most promising materials for gene delivery and gene therapy. In this study, we chemically modified a novel cross-linked PEI derivative PEI–Et through PEGylation, as PEGylation can reduce the cytotoxicity of PEI–Et by shielding the excess positive surface charge of PEI–Et. To screen out an optimal molar ratio of PEI–Et to PEG for efficient delivery of gene materials, we first synthesized three kinds of PEG–Et cationic polymers with different molar ratios (1 : 1, 2 : 1, 1 : 2) of PEI–Et to PEG, then we prepared and characterized the PEG–Et/DNA complexes and PEG–Et/siRNA complexes, and finally we tested the cytotoxicity and transfection efficiency of the PEG–Et/DNA complexes as well as the gene silencing efficiency of the PEG–Et/siRNA complexes. The results suggest that the gene vector PEG–Et 1 : 1 (with a 1 : 1 molar ratio of PEI–Et to PEG) was the best among the three types of PEG–Et (PEG–Et 1 : 1, PEG–Et 2 : 1, and PEG–Et 1 : 2) for condensing DNA and siRNA into nanoparticles, as it has significant lower cytotoxicity, higher gene transfection and siRNA silencing efficiency.

Nanoparticle-mediated RNA interference of angiotensinogen decreases blood pressure and improves myocardial remodeling in spontaneously hypertensive rats

Angiotensinogen (AGT) has been shown to have a role in cardiac hypertrophy, while depletion of the AGT gene in spontaneously hypertensive rats (SHR) has not been investigated. The present study investigated the effect of AGT knockdown on cardiac hypertrophy in SHR. For this, small hairpin (sh)RNAs were intravenously injected into SHRs, using a nanoparticle‑mediated transfection system. The experimental rats were divided into the following groups: a) Blank control with water treatment only, b) negative control with biscarbamate‑crosslinked Gal‑polyethylene glycol polyethylenimine nanoparticles (GPE)/negative shRNA, c) AGT‑RNA interference (RNAi) group with GPE/AGT‑shRNA, and 4) normotensive control using Wistar‑Kyoto rats (WKY) with water treatment. Three and five days following the first injection, the levels of hepatic AGT mRNA and AGT protein as well as plasma levels of AGT were markedly decreased in the AGT‑RNAi group (P<0.05). Furthermore, a significant decrease in systolic blood pressure (SBP), left ventricular weight to body weight ratio and heart weight to body weight ratio were observed in the AGT‑RNAi group compared with those in the control groups. The depletion of AGT in SHR led to a reduction in SBP by 30±4 mmHg, which was retained for >10 days. Cardiac hypertrophy was also significantly improved in AGT‑knockdown rats. In conclusion, the present study showed that AGT‑silencing had a significant inhibitory effect on hypertension and hypertensive‑induced cardiac hypertrophy in SHRs.