To optimize a receptor-mediated and cell-selective gene transfer with polyethyleneimine (PEI)-based vector, we synthesized three galactosylated PEIs (Gal-PEI) with different molecular weights (PEI(1800), PEI(10,000), and PEI(70,000)) and investigated their potential as a targetable vector to asialoglycoprotein receptor-positive cells. All PEI derivatives formed complexes with plasmid DNA (pDNA), whereas the particle size of the complex became smaller on increasing the molecular weight of PEI. Transfection efficiency in HepG2 cells with PEI was highest with PEI(1800); efficiency was next highest with PEI(10,000), although the cellular association was similar. After galactosylation, Gal(19)-PEI(10,000)/pDNA and Gal(120)-PEI(70,000)/pDNA showed considerable agglutination with a galactose-recognizing lectin, but Gal(9)-PEI(1800) did not, suggesting that galactose units on the Gal(9)-PEI(1800)-pDNA complex are not sufficiently available for recognition. Gal(19)-PEI(10,000)-pDNA and Gal(120)-PEI(70,000)-pDNA complexes showed galactose-inhibitable transgene expression in HepG2 cells. Transfection efficiency was greatest with Gal(19)-PEI(10,000)/pDNA, a result that highlights the importance of obtaining a balance between the cytotoxicity and the transfection activity, both of which are found to be a function of the molecular weight of PEI. After intraportal injection, however, Gal(153)-PEI(70,000)/pDNA having a low N/P ratio was most effective, suggesting that additional variables, such as the size of the complex, are important for in vivo gene transfer to hepatocytes.

No abstract

Poly(vinyl alcohol) (PVA) of various molecular weight (MW=10,560-116,600) was successfully labeled with fluorescein isothiocyanate isomer I (FITC) according to the method of de Belder and Granath. A high-performance size-exclusion chromatographic procedure was developed for the quantitative analysis of FITC-labeled poly(vinyl alcohol) (F-PVA) in biological samples. F-PVA (80 K) disappeared slowly from the blood circulation according to the first-order kinetics (t1/2=7 h) after intravenous injection to rats. A dose-independent behavior of F-PVA (80 K) was observed in the blood circulation, in the tissue distribution and in the urinary and fecal excretions. This suggested that PVAs are eliminated exclusively by the mechanisms that do not involve saturable transport processes. Furthermore, it was found that PVAs are very stable in the body because no degradation product was detected in the urine and feces. 125I-labeled poly(vinyl alcohol) (125I-PVA) was prepared by introducing tyramine residues to the hydroxyl groups of PVA molecules by the 1,1'-cabonyldiimidazole (CDI) activation method. 125I-PVA (80 K) was retained in the blood circulation for several days after intravenous injection to mice. Although the tissue distribution of PVAs was small, a significant accumulation into the liver and the spleen was observed. Fluorescence microscopic examination of paraffin section of the liver revealed that F-PVA (80 K) was endocytosed by the liver parenchymal cells. 125I-PVA (80 K) captured by liver was slowly transported via the bile canaliculi and gall bladder to the intestine and excreted in the feces. It was suggested, therefore, a long time is necessary for 125I-PVA (80 K) to be excreted perfectly from the body.

No abstract

Downregulation of lamin B1 has been recognized as a crucial step for development of full senescence. Accelerated cellular senescence linked to mechanistic target of rapamycin kinase (MTOR) signaling and accumulation of mitochondrial damage has been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. We hypothesized that lamin B1 protein levels are reduced in COPD lungs, contributing to the process of cigarette smoke (CS)–induced cellular senescence via dysregulation of MTOR and mitochondrial integrity. To illuminate the role of lamin B1 in COPD pathogenesis, lamin B1 protein levels, MTOR activation, mitochondrial mass, and cellular senescence were evaluated in CS extract (CSE)–treated human bronchial epithelial cells (HBEC), CS-exposed mice, and COPD lungs. We showed that lamin B1 was reduced by exposure to CSE and that autophagy was responsible for lamin B1 degradation in HBEC. Lamin B1 reduction was linked to MTOR activation through DEP domain–containing MTOR-interacting protein (DEPTOR) downregulation, resulting in accelerated cellular senescence. Aberrant MTOR activation was associated with increased mitochondrial mass, which can be attributed to peroxisome proliferator-activated receptor γ coactivator-1β–mediated mitochondrial biogenesis. CS-exposed mouse lungs and COPD lungs also showed reduced lamin B1 and DEPTOR protein levels, along with MTOR activation accompanied by increased mitochondrial mass and cellular senescence. Antidiabetic metformin prevented CSE-induced HBEC senescence and mitochondrial accumulation via increased DEPTOR expression. These findings suggest that lamin B1 reduction is not only a hallmark of lung aging but is also involved in the progression of cellular senescence during COPD pathogenesis through aberrant MTOR signaling.