Effect of molecular weight on the transfection efficiency of novel polyurethane as a biodegradable gene vector

New polyurethane 2-diethylaminoethylamine-polyurethane (LGEA-PU) containing poly(ethylene glycol) segments and tertiary amines was synthesized. LGEA-PU self-assembled readily with the plasmid DNA (pCMV-betagal) in HEPES buffer and was characterized by dynamic light scattering, zeta potential, atomic force microscopy, and XTT cell viability assays. To examine the effect of molecular weight of LGEA-PU systems on transfection, LGEA-PU systems of four different molecular weights (LGEA-PU99, LGEA-PU59, LGEA-PU24, and LGEA-PU7) were prepared. This study found that LGEA-PU99, LGEA-PU59, and LGEA-PU24 were able to bind plasmid DNA and yielded positively charged complexes with a nano-sized transfection (<200 nm). The LGEA-PU59/DNA complexes were able to transfect COS-7 cells in vitro with higher transfection efficiency than the other LGEA-PU systems. These results demonstrated that molecular weights of LGEA-PU systems had a significant effect on transferring ability, except for LGEA-PU99, which showed the strongest DNA condensation. Examination of the cytotoxicity of PEI and LGEA-PU systems revealed that LGEA-PU systems had lower cytotoxicity. In this article, LGEA-PU59 seemed to be a novel cationic polyurethane for gene delivery and an interesting candidate for further study.     

Platelet adsorption and hemolytic properties of liquid crystal/composite polymers

The aim of this study is to investigate how the presence of liquid crystal, cholesteryl oleyl carbonate, embedded into polymers (PMMA, Eb270, PU) affects the biocompatibility of composite membranes with human blood. The effects of different surface textures of composite membranes on platelet adhesion and platelet activation were evaluated as well. The adhesion and geometric deformation of platelets were demonstrated by SEM. The quantitative assay of platelet activation was determined by measuring the production of P-Selectin, and by measurement of the blood clotting index when PRP blood was incubated with pure polymer films and composite membranes. Moreover, the hemolysis studies on the damage to red blood cells were performed to gain information on the hemocompatibility of these biomaterials. The results showed that inclusion of cholesteryl oleyl carbonate (COC) embedded in composite membranes, improves their biocompatibility with respect to a substantial reduction of platelet adhesion and the controlled decrease of platelet activation. As the COC content of composite membranes was increased, the value of the blood clotting index increased and the production of P-Selectin decreased. The results also showed that the presence of COC resulted in a decrease of hemolysis ratios. Comparing among three different composite membranes, the best biocompatibility is achieved when PU/COC> or ==Eb270/COC>PMMA/COC. The in vitro studies performed in this work suggest that it may be reasonable to use liquid crystal COC as a mean of surface modification to improve the blood compatibility of biopolymers.     

Inhibition of cancer stem cell-like properties and reduced chemoradioresistance of glioblastoma using microRNA145 with cationic polyurethane-short branch PEI

Glioblastomas (GBMs) are the most common primary brain tumors with poor prognosis. CD133 has been considered a putative marker of cancer stem cells (CSCs) in malignant cancers, including GBMs. MicroRNAs (miRNAs), highly conserved small RNA molecules, may target oncogenes and have potential as a therapeutic strategy against cancer. However, the role of miRNAs in GBM-associated CSCs remains mostly unclear. In this study, our miRNA/mRNA-microarray and RT-PCR analysis showed that the expression of miR145 (a tumor-suppressive miRNA) is inversely correlated with the levels of Oct4 and Sox2 in GBM-CD133⁺ cells and malignant glioma specimens. We demonstrated that miR145 negatively regulates GBM tumorigenesis by targeting Oct4 and Sox2 in GBM-CD133⁺. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic-delivery vehicle, PU-PEI-mediated miR145 delivery to GBM-CD133⁺ significantly inhibited their tumorigenic and CSC-like abilities and facilitated their differentiation into CD133⁻-non-CSCs. Furthermore, PU-PEI-miR145-treated GBM-CD133⁺ effectively suppressed the expression of drug-resistance and anti-apoptotic genes and increased the sensitivity of the cells to radiation and temozolomide. Finally, the in vivo delivery of PU-PEI-miR145 alone significantly suppressed tumorigenesis with stemness, and synergistically improved the survival rate when used in combination with radiotherapy and temozolomide in orthotopic GBM-CD133⁺-transplanted immunocompromised mice. Therefore, PU-PEI-miR145 is a novel therapeutic approach for malignant brain tumors.     

Non-viral delivery of RNA interference targeting cancer cells in cancer gene therapy

RNA interference (RNAi) is a collection of small RNA-directed mechanisms that result in sequence-specific inhibition of gene expression. RNAi delivery has demonstrated promising efficacy in the treatment of genetic disorders in cancer. Although viral vectors are currently the most efficient systems for gene therapy, potent immunogenicity, mutagenesis, and the biohazards of viral vectors remain their major risks. Various non-viral delivery vectors have been developed to provide a safer approach for gene delivery, including polymers, peptides, liposomes, and nanoparticles. However, some concerns and challenges of these non-viral gene delivery approaches remain to be overcome. In this review, we summarize the recent progress in the development of non-viral systems delivering RNAi and the currently available preclinical and clinical data, and discuss the challenges and future directions in cancer therapy.     

Potential protective effect of fresh grown unicellular green algae component (resilient factor) against PMA- and UVB-induced MMP1 expression in skin fibroblasts

Solar UV radiation damages human skin, affecting skin tone and resiliency, and leading to premature ageing (photoageing). Skin damage by oxidants may lead to activation of PKC, thus increasing matrix metalloproteinase (MMPs) expression and collagen degradation. Administration of Chlorella has been shown to play some biochemical functions as well as in vitro inhibition of MMP1 activity. MMP1 secretion was evaluated following PMA treatment or UVB irradiation in the presence of Resilient Factor (RF, aqueous extract fraction of Chlorella), vitamin C, or vitamin E in human skin fibroblasts. Expression levels of MMP1 and elastin protein and of MMP1, TIMP1, and pro-collagen mRNA were also investigated. PMA-induced MMP1 production, protein, and gene expression were suppressed in the presence of RF. Elastin protein diminished after UVB exposure and RF treatment appeared able to counteract the effect of UVB irradiation. Our results also suggest that RF may increase pro-collagen mRNA expression following UVB exposure. This study shows that application of RF prevents MMP1 production via the inhibition of protein and gene expression. In addition, RF prevents the UVB-suppressed elastin protein and pro-collagen gene expression. These findings indicate that RF may exert a protective effect against UVB irradiation-induced damage in the skin.     

Effect of Size and Serum Proteins on Transfection Efficiency of Poly ((2-dimethylamino)ethyl Methacrylate)-Plasmid Nanoparticles

Purpose. The aim of this study was to gain insight into the relation between the physical characteristics of particles formed by a plasmid and a synthetic cationic polymer (poly(2-dimethylamino)ethyl methacrylate, PDMAEMA) and their transfection efficiency. Methods. The PDMAEMA-plasmid particles were characterized by dynamic light scattering (size) and electrophoretic mobility measurements (charge). The transfection efficiency was evaluated in cell culture (COS-7 cells) using a pCMV-lacZ plasmid coding for -galactosidase as a reporter gene. Results. It was shown that the optimal transfection efficiency was found at a PDMAEMA-plasmid ratio of 3 (w/w), yielding stable and rather homogeneous particles (diameter 0.15 µm) with a narrow size distribution and a slightly positive charge. Particles prepared at lower weight ratios, showed a reduced transfection efficiency and were unstable in time as demonstrated by DLS measurements. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. Interestingly, the transfection efficiency of the particles was not affected by the presence of serum proteins. Conclusions. PDMAEMA is an interesting vector for the design of in vivo and ex vivo gene transfection systems.     

Long Term Stability of Poly((2-dimethylamino)ethyl Methacrylate)-Based Gene Delivery Systems

Purpose. To study the stability of polymer-plasmid complexes (polyplexes) both as an aqueous dispersion and in their lyophilized form. Methods. The characteristics of the polyplexes (size, charge and transfection potential) were monitored at different temperatures. Moreover, we studied possible changes in the secondary and tertiary structure of the plasmid by agarose gel electrophoresis and by CD spectroscopy to gain insight into the mechanism of polyplex degradation. Results. The polyplexes preserved almost their full transfection potential after aging in an aqueous solution of 20 mM Hepes (pH 7.4) containing 10% sucrose at 4 and 20°C for 10 months. On the other hand, the polyplexes aged at 40°C were rather unstable and lost their transfection capability with a half-life of around 2 months. During storage, conformational changes in the secondary and tertiary structure of DNA were observed. When naked plasmid DNA was aged at 40°C as an aqueous solution and complexed with polymer just before the transfection experiment, a slower drop in its transfection capability was observed. The freeze-dried polyplexes using sucrose as lyoprotectant almost fully retained their transfection efficiency, even when aged at 40°C for 10 months. Conclusions. This study provides information about polyplex stability in aqueous dispersions on storage and demonstrates that freeze-drying is an excellent method to ensure long term stability.     

Freeze-Drying of Poly((2-dimethylamino)ethyl Methacrylate)-Based Gene Delivery Systems

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