Reinforcement versus Fluidization in Cytoskeletal Mechanoresponsiveness

Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment

Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses

<p>Abstract</p> <p>Background</p> <p>Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA.</p> <p>Results</p> <p>In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed <it>in vitro</it>. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (<it>P </it>< 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220⁺cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-A^d) were increased on CD11c⁺dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice.</p> <p>Conclusion</p> <p>These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.</p

Reinforcement versus Fluidization in Cytoskeletal Mechanoresponsiveness

Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment

폐암 치료를 위한 DNA 와 siRNA 전달체로서 분해성 고분자의 합성과 평가

Thesis(doctors) --서울대학교 대학원 :농생명공학부, 2009.8.Docto

Challenges for cell-based medicinal products from a pharmaceutical product perspective

Advanced therapy medicinal products (ATMPs), such as somatic cell-therapy medicinal products or tissue-engineered products for human use, offer new and potentially curative opportunities to treat yet untreatable diseases or disorders. For cell-therapy medicinal products (CBMPs), multiple stability and quality challenges exist and relate to the cellular composition and unstable nature of these parenteral preparations. It is the aim of this review to discuss open questions and problems associated with the development, manufacturing and testing of CBMPs from a pharmaceutical drug product perspective. This includes safety, storage and handling, particulates, the choice of container closure systems and integrity. Analytical methods commonly used to evaluate the quality of the final CBMP product to ensure patient's safety will be discussed. Particulate contamination in final products deserve special attention since CBMPs cannot be sterile filtered. Visible and sub-visible particles may represent environmental contaminations or may form during storage. They may be introduced from processing materials such as single use product contact materials, ancillary materials, or any components such as primary packaging used for the final product. Currently available analytical methods for detecting particulates may not be easily applicable to CBMPs due to their inherent particulate nature and appearance

Subvisible Particulate Contamination in Cell Therapy Products-Can We Distinguish?

Cell therapy products represent an exciting new class of medicinal products, which must be parenterally administered. Thus, compliance with parenteral preparation guidelines is required. One requirement for parenteral products is the characterization of particle contaminations. As cell-based products are turbid suspensions, containing particles, the cells, characterization and control of foreign particle impurities remain a challenge. Within this study, we evaluated a flow imaging microscopy method for the detection and characterization of subvisible particle contaminations in cell-based products. We found that flow imaging microscopy is a potential method where subvisible particle contaminations can be differentiated from the cells in cell therapy products

Biodegradable Nano-Polymeric System for Efficient Akt1 siRNA Delivery

Biodegradable nano-polymeric carrier composed of polycaprolactone (PCL) and polyethylenimine (PEI) (BNPP) was successfully synthesized for the delivery of sh/siRNA in lung cancer cells. BNPP efficiently and safely delivered siRNA in lung cancer cells. BNPP-delivered Akt1 siRNA silenced Akt1 protein, and reduced the cancer cell survival, proliferation, malignancy and metastasis

Bioreducible polymers for efficient gene and siRNA delivery

Bioreducible disulfide linkage-employing drug conjugate has already been approved for drug delivery application, and also has shown immense potential in gene and siRNA transfection. This paper will focus on the recent developments in bioreducible polymeric systems for gene and siRNA delivery application, and will discuss the advantages and challenges associated with reducible polymeric carriers

Poly(beta-amino ester) as a carrier for si/shRNA delivery in lung cancer cells

Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNA interference (RNAi) studies. In the present study, we evaluated biodegradable poly(beta-amino ester) (PAE) carrier composed of low molecular weight polyethylenimine and poly(ethylene glycol) for si/shRNA delivery in lung cancer cells. PAE carrier successfully delivered EGFP (enhanced green fluorescence protein) siRNA (siGFP) and silenced EGFP expression. The silencing achieved with PAE carrier was found to be nearly 1.5 times superior and safer than standard PEI25K. Also, our PAE carrier exhibited superior Akt1 shRNA delivery (shAkt) and thereby silenced oncoprotein Akt1 efficiently. PAE-shAkt mediated Akt1 knock-down hindered cancer cell growth in Akt1 specific manner. Superior shAkt delivery and low cytotoxicity of PAE carrier promoted Akt1 knock-down specific apoptosis, while low delivery efficiency and high cytotoxicity of PEI25K carrier mainly exhibited undesirable necrosis. Moreover, basic cancer properties like cell proliferation, malignancy and metastasis were reduced more efficiently using PAE-shAkt system. These findings demonstrated the potential of PAE as an alternative to PEI25K in si/shRNA-based RNAi studies. (c) 2008 Elsevier Ltd. All rights reserved

The suppression of lung tumorigenesis by aerosol-delivered folate-chitosan-graft-polyethylenimine/Akt1 shRNA complexes through the Akt signaling pathway

RNA interference (RNAi) represents a promising new approach to the inhibition of gene expression in vitro and in vivo, and has therapeutic potential for human diseases. Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNAi studies. Here we report the development of a new polymeric gene carrier for cancer cell-targeting, designed to enhance the intracellular delivery of shRNA and reduce cytotoxicity. Folate-chitosan-graft-polyethylenimine (FC-g-PEI) copolymer was prepared by an imine reaction between periodate-oxidized folate-chitosan (FC) and low molecular weight polyethylenimine (PEI). FC-g-PEI copolymer was investigated as a potential cancer cell-targeting gene carrier. The composition of FC-g-PEI was characterized using (1)H nuclear magnetic resonance ((1)H NMR), and particle size and zeta potential of FC-g-PEI/shRNA complexes were measured using dynamic light scattering (DLS). FC-g-PEI showed good shRNA condensation ability and high protection of shRNA from nuclease attack. it also exhibited lower cytotoxicity compared to PEI 25K control, and showed good cancer cell-targeting ability. Furthermore, aerosol delivery of FC-g-PEI/Akt1 shRNA complexes suppressed lung tumorigenesis in a urethane-induced lung cancer model mouse through the Akt signaling pathway. Together, these results suggest that FC-g-PEI may be useful for shRNA-based gene therapy. (C) 2009 Elsevier Ltd. All rights reserved