Glucose-Responsive Gene Delivery at Physiological pH through Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction

We report a physiologically stable and cytocompatible glucose-responsive nonviral gene delivery system made up of boronate functionalized polymeric material. Herein, we utilize boronate cis-diol interactions to develop a glucose-responsive submicron particle (SMP) system. The stability of the boronate interaction at a physiological pH was achieved by copolymerization of dimethyl aminoethyl methacrylate (DMAEMA) with acrylamidophenylboronic acid (AAPBA) and the formation of a complex with polyvinylalcohol (PVA) which is governed by cis-diol interactions. The shift in hydrodynamic diameter of SMPs was observed and correlated with increasing glucose concentrations at a physiological pH. Optimal transfection was observed for a 5 µg dose of the gaussia luciferase reporter gene in NIH3T3 cells without any adverse effect on cellular viability. The destabilization of the AAPBA–PVA complex by interacting with glucose allowed the release of encapsulated bovine serum albumin (BSA) in a glucose-responsive manner. In total, 95% of BSA was released from SMPs at a 50 mM glucose concentration after 72 h. A two-fold increase in transfection was observed in 50 mM glucose compared to that of 10 mM glucose.

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Evaluation by Fluorescence Resonance Energy Transfer of the Stability of Nonviral Gene Delivery Vectors under Physiological Conditions

Biomacromolecules ◽

10.1021/bm025527d ◽

2002 ◽

Vol 3 (4) ◽

pp. 841-845 ◽

Cited By ~ 67

Author(s):

Keiji Itaka ◽

Atsushi Harada ◽

Kozo Nakamura ◽

Hiroshi Kawaguchi ◽

Kazunori Kataoka

Keyword(s):

Energy Transfer ◽

Gene Delivery ◽

Fluorescence Resonance Energy Transfer ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Nonviral Gene Delivery ◽

Physiological Conditions ◽

Gene Delivery Vectors ◽

Fluorescence Resonance ◽

The Stability

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ELK4 promotes the development of gastric cancer by inducing M2 polarization of macrophages through regulation of the KDM5A-PJA2-KSR1 axis

Journal of Translational Medicine ◽

10.1186/s12967-021-02915-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Lei Zheng ◽

Hongmei Xu ◽

Ya Di ◽

Lanlan Chen ◽

Jiao Liu ◽

...

Keyword(s):

Gastric Cancer ◽

Transcriptional Activation ◽

Luciferase Reporter ◽

Loss Of Function ◽

Luciferase Reporter Gene ◽

M2 Polarization ◽

Lysine Specific Demethylase ◽

Dependent Inhibition ◽

Kinase Suppressor Of Ras ◽

The Stability

Abstract Background We tried to elaborate the molecular mechanism of ETS-like transcription factor 4 (ELK4) affecting gastric cancer (GC) progression through M2 polarization of macrophages mediated by lysine-specific demethylase 5A (KDM5A)-Praja2 (PJA2)-kinase suppressor of ras 1 (KSR1) axis. Methods GC expression dataset was obtained from GEO database, and the downstream regulatory mechanism of ELK4 was predicted. Tumor-associated macrophages (TAMs) were isolated from GC tissues. The interaction among ELK4, KDM5A, PJA2 and KSR1 was analyzed by dual luciferase reporter gene, ChIP and Co-IP assays. The stability of KSR1 protein was detected by cycloheximide (CHX) treatment. After TAMs were co-cultured with HGC-27 cells, HGC-27 cell biological processes were assessed through gain- and loss-of function assays. Tumorigenicity was detected by tumorigenicity test in nude mice. Results In GC and TAMs, ELK4, KDM5A and KSR1 were highly expressed, while PJA2 was lowly expressed. M2 polarization of macrophages promoted the development of GC. ELK4 activated KDM5A by transcription and promoted macrophage M2 polarization. KDM5A inhibited the expression of PJA2 by removing H3K4me3 of PJA2 promoter, which promoted M2 polarization of macrophages. PJA2 reduced KSR1 by ubiquitination. ELK4 promoted the proliferative, migrative and invasive potentials of GC cells as well as the growth of GC xenografts by regulating KSR1. Conclusion ELK4 may reduce the PJA2-dependent inhibition of KSR1 by transcriptional activation of KDM5A to promote M2 polarization of macrophages, thus promoting the development of GC.

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Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts

Cells ◽

10.3390/cells8121532 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1532 ◽

Cited By ~ 1

Author(s):

Ning Li ◽

Joachim F. Uhrig ◽

Corinna Thurow ◽

Li-Jun Huang ◽

Christiane Gatz

Keyword(s):

Signaling Pathway ◽

Function Analysis ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Protein Levels ◽

Activation Of Transcription ◽

Subsequent Degradation ◽

The Stability ◽

F Box Protein ◽

Ja Signaling

The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aos coi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.

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Comparative Investigation of Polymeric and Nanoparticle Vehicles for Transgene Delivery

Nano LIFE ◽

10.1142/s1793984416410014 ◽

2016 ◽

Vol 06 (02) ◽

pp. 1641001 ◽

Cited By ~ 1

Author(s):

James Ramos ◽

Kaushal Rege

Keyword(s):

Gene Delivery ◽

Transgene Expression ◽

Comparative Investigation ◽

Nonviral Gene Delivery ◽

Luciferase Expression ◽

Hydrodynamic Diameter ◽

Nucleic Acid Delivery ◽

Polymeric Systems ◽

Transgene Delivery

Effective design of nanoparticle systems can have a significant impact on therapeutic delivery. Physicochemical factors including size, shape and surface chemistry of nanoparticles can play a significant role in determining the efficacy of drug and gene delivery to cells. Polymeric as well as inorganic nanoparticle systems have been investigated as vehicles for nonviral gene delivery of transgenes. However, a head-to-head comparison of these different systems is largely lacking. In this study, we compare three related delivery systems, polymer, polymer-templated gold nanospheres and polymer-coated gold nanorods, for their respective in vitro transgene expression efficacies. Significant differences were seen in the hydrodynamic diameter and zeta potential for each of these different vehicles. Nevertheless, transgene (luciferase) expression efficacies and cytotoxicities were found to be similar for these three vehicles under different conditions. Our results indicate that polymeric systems can be used for synthesis or modification of nanoparticle systems with negligible variations in their transgene expression efficacies. This can have significant implications for generating polymer-nanoparticle theranostic systems that maintain therapeutic (e.g., nucleic acid) delivery efficacies, while also possessing additional functionalities, including

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DNA Melting Temperature Assay for Assessing the Stability of DNA Polyplexes Intended for Nonviral Gene Delivery

Langmuir ◽

10.1021/la201803c ◽

2011 ◽

Vol 27 (19) ◽

pp. 12042-12051 ◽

Cited By ~ 20

Author(s):

Anja Schallon ◽

Christopher V. Synatschke ◽

Dmitry V. Pergushov ◽

Valérie Jérôme ◽

Axel H. E. Müller ◽

...

Keyword(s):

Gene Delivery ◽

Melting Temperature ◽

Nonviral Gene Delivery ◽

Dna Melting ◽

The Stability

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Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue

Journal of Virology ◽

10.1128/jvi.72.11.8568-8577.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8568-8577 ◽

Cited By ~ 310

Author(s):

Dongsheng Duan ◽

Prerna Sharma ◽

Jusan Yang ◽

Yongping Yue ◽

Lorita Dudus ◽

...

Keyword(s):

Muscle Tissue ◽

Structural Characteristics ◽

Shuttle Vector ◽

Fold Increase ◽

Structural Features ◽

Nonviral Gene Delivery ◽

Adeno Associated Virus ◽

Great Utility ◽

The Stability

ABSTRACT Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.

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Gaussia Luciferase Reporter Assay for Assessment of Gene Delivery Systems in Vivo

Chinese Medical Sciences Journal ◽

10.1016/s1001-9294(10)60029-6 ◽

2010 ◽

Vol 25 (2) ◽

pp. 95-99 ◽

Cited By ~ 4

Author(s):

Feng Chen ◽

Zhen Xu ◽

Jing Lu ◽

Xiang Lü ◽

Wen-li Mu ◽

...

Keyword(s):

Gene Delivery ◽

Delivery Systems ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Gaussia Luciferase

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Efficient Gene Delivery Using Anionic Liposome-Complexed Polyplexes (LPDII)

Bioscience Reports ◽

10.1023/a:1010338219401 ◽

2000 ◽

Vol 20 (5) ◽

pp. 419-432 ◽

Cited By ~ 39

Author(s):

Wenjin Guo ◽

Robert J. Lee

Keyword(s):

Gene Delivery ◽

Mammalian Cells ◽

High Efficiency ◽

Transfection Efficiency ◽

Cationic Lipids ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Efficient Gene ◽

Anionic Liposomes ◽

Cholesteryl Hemisuccinate

Synthetic gene transfer vectors based on polyplexes complexed to anionic liposomes (LPDII vectors) were characterized for their transfection efficiency in cultured mammalian cells. The effects of polycation to DNA ratio, lipid to DNA ratio, choice of polycation and lipid composition were systematically evaluated in human oral carcinoma KB cells, using a luciferase reporter gene. For LPDII formulations containing poly-L-lysine and dioeoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) anionic liposomes, at a constant lipid to DNA ratio, an increase in the polycation/DNA (N/P) ratio resulted in an increase in transfection activity. Meanwhile, the optimal lipid to DNA ratio for efficient gene delivery was influenced by the N/P ratio used, and was increased at higher N/P ratios. For the DNA condensing agent, poly-L-lysine could be replaced by polyethylenimine (PEI) as the DNA condensing agent in the formulations. For the lipidic components, CHEMS could be replaced by other anioniclipids including oleic acid, dicetylphosphate and phosphatidylserine, but DOPE, a fusogenic helper lipid, could not be replaced by dioleolyphosphatidylcholine. LPDII formulation showed significantly less cytotoxicity compared to the commonly used cationic lipsomes or PEI mediated transfection and several cell lines were transfected with high efficiency. LPDII vectors avoid the use of toxic cationic lipids and may have potential application in gene therapy.

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A luciferase-engineered cell line for study of cAMP regulation in endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.1.c75 ◽

1998 ◽

Vol 275 (1) ◽

pp. C75-C81 ◽

Cited By ~ 28

Author(s):

J. Xavier-Neto ◽

A. C. Pereira ◽

A. H. Motoyama ◽

J. E. Krieger

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Line ◽

Fold Increase ◽

Positive Control ◽

Luciferase Reporter ◽

Luciferase Expression ◽

Luciferase Reporter Gene ◽

Aortic Endothelial Cells ◽

Endothelial Cell Line

cAREL is a cAMP-responsive endothelial cell line carrying a luciferase reporter gene introduced by stable transfection of a luciferase enhancer trap into rabbit aortic endothelial cells. Luciferase gene expression in cAREL was stimulated 233-fold by 8-BrcAMP. Treatment with the β-adrenoceptor agonist isoproterenol induced a 7.0-fold increase in luciferase expression, which was partially blocked by either β1- or β2-adrenoceptor antagonists and totally blocked by propranolol and by a combination of β1- plus β2-adrenoceptor antagonists. Receptor stimulation was mimicked by cholera toxin, forskolin, 8-BrcAMP, and isobutylmethylxanthine but not by 8BrcGMP, dexamethasone, or phorbol 12-myristate 13-acetate. Stimulation by isoproterenol was completely blocked by H-89, a protein kinase A inhibitor. cAREL was also stimulated by A-23187, and this effect was abrogated by EGTA and H-89. cAREL is the first cAMP-sensitive endothelial cell line described, and it can be useful as a positive control, as a model for cAMP regulation, as a background to genetic introduction of receptors, as an indicator of intracellular pathway activation, and as a tool to investigate cAMP effects on other signaling pathways.

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