Immune complexome analysis of a rich variety of serum immune complexes identifies disease-characteristic immune complex antigens in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular endothelial dysfunction and skin fibrosis. Recently, the presence and pathogenic role of immune complexes (ICs) of SSc patients were reported. However, the identities of antigens in these ICs are unknown. Therefore, we examined ICs in the serum of SSc patients to elucidate SSc pathogenesis. In this study, IC concentrations in serum samples from SSc and systemic lupus erythematosus (SLE) patients were measured by C1q enzyme-linked immunosorbent assays; immune complex analysis was used for comprehensive identification and comparison of antigens incorporated into ICs (IC-antigens). The expression patterns of SSc-specific IC-antigens in skin sections were investigated by immunohistochemistry. Compared with SLE patients who developed disease because of IC deposition, SSc patients had a greater number of IC-antigens and a smaller difference in IC concentrations, suggesting that SSc pathogenesis is affected by the proteins present in ICs. In contrast, the IC concentration and number of IC-antigens did not significantly differ according to the clinical phenotype of SSc. We identified 478 IC-antigens in SSc patients, including multiple RNAP II-associated proteins that were targeted by antibodies previously associated with SSc pathogenesis. The most frequently detected RNAP II-associated protein, RNA polymerase II transcription subunit 30 (MED30), was strongly expressed at lesion sites and reportedly regulates endothelial differentiation. Therefore, increased expression of MED30 in lesions may have an antigenic effect, and MED30 function may be impaired or inhibited by IC formation. RNAP II-associated proteins may SSc pathogenesis through mechanisms such as the MED30 pathway.

Development of a Two-Layered Sheet Formulation of 5-Fluorouracil for Application to Rat's Livers to Ensure Controlled Release and Local Drug Disposition.

This study aimed to develop a new and effective application form for the liver surface. We designed a two-layered sheet for the controlled release and local disposition of the anticancer drug, 5-fluorouracil (5-FU), without leakage into the peritoneal cavity. We employed poly(lactic-co-glycolic acid) (PLGA) and hydroxypropyl cellulose (HPC) to form two-layered sheets by attaching a cover sheet and a drug-containing sheet. The prepared two-layered sheets released 5-FU constantly for up to 14 d without any significant leakage from the cover side in vitro. Furthermore, we have applied sheets containing 5-FU to the rat liver surface in vivo. Notably, 5-FU could be detected in the liver attachment region even 28 d after application. The distribution ratio of 5-FU in the attachment region compared to the other liver lobes varied among the sheet formulations with different additive HPC compositions. The area under the liver concentration-time curve (AUC) of 5-FU in the attachment region from 0 to 28 d was the highest in the case of HPC 2% (w/w). This is probably due to the enhanced 5-FU released amount and controlled absorption rate from the liver surface by released HPC. No critical toxic effects were evident by the application of the two-layered sheets from the body weight change and alanine aminotransferase/aspartate aminotransferase (ALT/AST) activities. Consequently, the possible advantage of the two-layered sheets for prolonged retention of a drug in a specific region in the liver was clarified.

Influence of Liver Intoxication by Carbon Tetrachloride or D-Galactosamine on Absorption of Fluorescein Isothiocyanate-Dextran-10 and Other Marker Compounds with Different Molecular Weights from the Rat Liver Surface.

We examined the influence of liver disease on the absorption from the liver surface of fluorescein isothiocyanate (FITC)-dextran 10 (FD-10, MW: 11000) and several marker compounds with different molecular weights. The purpose of this study was to determine the feasibility of liver surface application of macromolecular compounds in the disease state. We used male Wistar rats treated with carbon tetrachloride (CCl4) or D-galactosamine (GAL). FD-10 and other marker compounds were applied to the liver surface using a cylindrical diffusion cell in liver-intoxicated rats. The blood, bile, urine, and the remaining solution in the diffusion cell were collected for assay. FD-10 was absorbed by first-order kinetics from the liver surface in the liver-intoxicated rat models. The calculated rate constant ka values in the normal, CCl4 and GAL groups were 0.000965, 0.00125 and 0.00104 min-1, respectively. Increased absorption of FITC-dextrans in the liver-intoxicated rats was observed. In both CCl4 and GAL groups, an inverse relationship was observed between the molecular weight and ka from the rat liver surface of the marker compounds. The limits of the molecular weight absorbed from the liver surface were extrapolated to be 71200, 135000, and 105000 in the normal, CCl4, and GAL groups, respectively. In conclusion, increased absorbability from the rat liver surface indicates that liver surface application for liver targeting of macromolecules in the diseased state is indeed feasible. Therefore, our findings can support further research on liver surface application of drugs under liver disease.

Effect of Chronic Kidney Disease on Hepatic Clearance of Drugs in Rats.

The pharmacokinetics of some hepatically cleared drugs have been reported to fluctuate in patients with renal impairment, but the definitive factors have not been clarified. We compared the pharmacokinetics of some drugs with different hepatic elimination processes in a chronic kidney disease (CKD) rat model, to optimize their administration during kidney injury. We chose indocyanine green (ICG), midazolam (MDZ), and acetaminophen (APAP) as reference drugs to determine changes in hepatic clearance pathways in presence of CKD. Drugs were intravenously administered via the jugular vein to the CKD model rats, previously established by adenine administration, and then, blood, bile, and urine samples were collected. The plasma concentration of ICG, which is eliminated into the bile without biotransformation, increased; and its total body clearance (CLtot) significantly decreased in the CKD group compared to the control group. Moreover, the plasma concentrations of MDZ and APAP, metabolized in the liver by CYP3A and Ugt1a6 enzymes, respectively, were higher in the CKD group than in the control group. The biliary clearances of APAP and its derivative APAP-glucuronide increased in the CKD group, whereas their renal clearances were markedly decreased with respect to those in the control group. Altogether, plasma concentrations of some hepatically eliminated drugs increased in the CKD rat model, but depending on their pharmacokinetic characteristics. This study provides useful information for optimizing the administration of some hepatically cleared drugs in CKD patients.

Evaluation of mRNA expression of drug-metabolizing enzymes in acetaminophen-induced hepatotoxicity using a three-dimensional hepatocyte culture system.

1. The expression and activity of drug-metabolizing enzymes are known to affect the pharmacokinetics of drugs metabolized in the liver. Here, we assessed the effect of acetaminophen (APAP)-induced hepatotoxicity on the mRNA expression of drug-metabolizing enzymes and elucidated the underlying mechanism using three-dimensional (3D) cultures of HepG2 cells.2. 3D culture cells enabled us to establish an in vitro model of APAP-induced hepatotoxicity which showed the increase in N-acetyl-p-benzoquinone imine production, reactive oxygen species (ROS) generation and cellular injury.3. In this 3D culture model, APAP treatment significantly increased the mRNA expression of drug-metabolizing enzymes (cytochrome P450 [CYP]3A4, CYP2E1 and UDP-glucuronosyltransferase 1A6) and their nuclear receptors (pregnane X receptor and constitutive androstane receptor) compared with untreated cells. Treatment with N-acetylcysteine, a therapeutic agent for APAP-induced hepatotoxicity, suppressed these increases. In addition, the mRNA expression of drug-metabolizing enzymes and nuclear receptors were elevated depending on the concentration of H2O2, one of ROS involved in the development of APAP-induced hepatotoxicity. The mRNA expression of nuclear receptors increased before that of drug-metabolizing enzymes.4. In conclusion, ROS may induce the mRNA expression of nuclear receptors and promote the transcription of drug-metabolizing enzymes in the in vitro model of APAP-induced hepatotoxicity.

Influence of vasomodulators and tumor transplantation on the disposition of 5-fluorouracil after application to the liver surface in rats.

This study investigated the effect of epinephrine (a vasoconstrictor) and hydralazine (a vasodilator) on the hepatic disposition of 5-fluorouracil (5-FU) after application to the surface of the liver in rats. Normal livers were compared with a Walker 256 carcinoma cell tumor model. A cylindrical diffusion cell was attached to the liver surface. 5-Fluorouracil was added into the diffusion cell in combination with vasomodulators or after pretreatment with epinephrine. After selected treatment times, the 5-FU concentrations were assayed at three sites in the excised livers. The 5-FU concentration in the region under the cell attachment site (site 1) was significantly higher after concomitant application of 5-FU and epinephrine, compared with 5-FU alone, and increased in an epinephrine dose-dependent manner. On the other hand, preferential distribution of 5-FU at site 1 was not seen when applied in combination with hydralazine. After 10 min of epinephrine pretreatment, the concentration of 5-FU at site 1 was approximately two times higher than that for the control. Furthermore, the 5-FU concentration at site 1 of the tumor model was greatly increased compared with the normal liver. These results suggest that application of epinephrine to the liver surface might enhance the accumulation of 5-FU at the desired target site.

Evaluation for Peritoneal Injury at an Early Stage Using Dual Macromolecular Markers.

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, making continuation of PD difficult. Therefore, it is necessary to evaluate peritoneal injury at an early stage and develop appropriate therapies. The aims of the present study were to evaluate peritoneal injury at an early stage and assess a drug for prevention of peritoneal injury using our previously developed novel evaluation method. Peritoneal injury was induced in model animals by intraperitoneal injection of methylglyoxal (MGO) for 1 to 5 consecutive days or chlorhexidine digluconate (CG) for 1 to 14 consecutive days. Tetramethylrhodamine-dextran (RD)-10 and fluorescein isothiocyanate-dextran (FD)-2000 were then injected into the peritoneal cavity and recovered after 120 min to evaluate peritoneal injury. The ratio of the concentration of RD-10 to FD-2000 (RD-10/FD-2000 ratio) significantly decreased in animals that had been treated with MGO or CG for 1 d. Moreover, the RD-10/FD-2000 ratio significantly increased in CG- and thalidomide-treated animals. The RD-10/FD-2000 ratio can be used to evaluate peritoneal injury at an early stage and assess the drug efficacy of thalidomide for prevention of peritoneal injury. This study will contribute to the development of therapeutic treatments for peritoneal injury.

Effect of Metabolic Inhibitors on the Hepatic Disposition of 5-Fluorouracil after Application to the Rat Liver Surface.

We evaluated the effects of 5-fluorouracil (5-FU) metabolic inhibitors, gimeracil or uridine, on the hepatic disposition of 5-FU after application to the liver surface in rats, aiming to enhance the availability of 5-FU in the liver. 5-FU solution with or without metabolic inhibitors was applied to the rat liver surface using a cylindrical diffusion cell. The liver, blood and the remaining solution in the diffusion cell were collected at specified times, and assayed for 5-FU content. 5-FU absorption properties were not altered by addition of gimeracil and uridine. The 5-FU concentration in the diffusion cell attachment site of the rat liver (site 1) at 0.1-0.4 M ratios of gimeracil to 5-FU was significantly higher than that of the control. On the contrary, the addition of uridine did not increase the 5-FU concentration at site 1. At a 0.1 M ratio of gimeracil to 5-FU, the maximum 5-FU plasma concentration was the lowest, and the area under the 5-FU concentration-time curve at site 1 was 3.4 times greater than that of the control. We demonstrated that applying 5-FU with gimeracil to the rat liver surface could increase the availability of 5-FU in the liver.

Evaluation of hypothermia on the in vitro metabolism and binding and in vivo disposition of midazolam in rats.

The effect of hypothermia on the in vivo pharmacokinetics of midazolam was evaluated, with a focus on altered metabolism in the liver and binding to serum proteins. Rat primary hepatocytes were incubated with midazolam (which is metabolized mainly by CYP3A2) at 37, 32 or 28 °C. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of midazolam were estimated using the Michaelis-Menten equation. The Km of CYP3A2 midazolam remained unchanged, but the Vmax decreased at 28 °C. In rats, whose temperature was maintained at 37, 32 or 28 °C by a heat lamp or ice pack, the plasma concentrations of midazolam were higher, whereas those in the brain and liver were unchanged at 28 °C. The tissue/plasma concentration ratios were, however, increased significantly. The unbound fraction of midazolam in serum at 28 °C was half that at 37 °C. These pharmacokinetic changes associated with hypothermic conditions were due to reductions in CYP3A2 activity and protein binding.

Novel diagnostic method of peritoneal injury using dual macromolecular markers.

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, which makes continuation of PD difficult. Moreover, the progression of peritoneal injury causes complications and poor prognosis. Since therapeutic treatments for peritoneal injury during PD have yet to be established, it is important to diagnose peritoneal injury as early as possible. The aim of this study was to develop a method of monitoring peritoneal function to diagnose peritoneal injury. Model rats of peritoneal injury were prepared by intraperitoneal injection of methylglyoxal (MGO) for five consecutive days. Then, marker substances of various molecular weights (phenolsulfonphthalein, fluorescein isothiocyanate-dextran (FD)-10, FD-40, FD-70, FD-2000 or tetramethylrhodamine-dextran (RD)-10) were injected into the peritoneal cavity. At 120 min after injection, the remaining amounts of all marker substances were significantly decreased in the MGO-treated rats compared with those in the vehicle-treated rats. Molecular weight dependence of the peritoneal permeability was observed. A substance with a molecular weight of approximately 10000 was found to be suitable to diagnose peritoneal injury. Moreover, coadministration of RD-10 with FD-2000 enabled us to monitor enhanced peritoneal permeability and the transfer of water simultaneously, without the recovery of whole PD fluid, even in the case of different ultrafiltration volumes. We demonstrated the usefulness of administering substances to evaluate peritoneal permeability and the transfer of water simultaneously to diagnose peritoneal injury. This study should be valuable for safe and effective PD.

One-Step Formation Method of Plasmid DNA-Loaded, Extracellular Vesicles-Mimicking Lipid Nanoparticles Based on Nucleic Acids Dilution-Induced Assembly.

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations.

Interaction of γ-Polyglutamic Acid/Polyethyleneimine/Plasmid DNA Ternary Complexes with Serum Components Plays a Crucial Role in Transfection in Mice.

Typical examples of non-viral vectors are binary complexes of plasmid DNA with cationic polymers such as polyethyleneimine (PEI). However, problems such as cytotoxicity and hemagglutination, owing to their positively charged surfaces, hinder their in vivo use. Coating binary complexes with anionic polymers, such as γ-polyglutamic acid (γ-PGA), can prevent cytotoxicity and hemagglutination. However, the role of interactions between these complexes and serum components in in vivo gene transfer remains unclear. In this study, we analyzed the contribution of serum components to in vivo gene transfer using PEI/plasmid DNA binary complexes and γ-PGA/PEI/plasmid DNA ternary complexes. In binary complexes, heat-labile components in the serum greatly contribute to the hepatic and splenic gene expression of the luciferase gene. In contrast, serum albumin and salts affected the hepatic and splenic gene expression in the ternary complexes. Changes in physicochemical characteristics, such as increased particle size and decreased absolute values of ζ-potential, might be involved in the enhanced gene expression. These findings would contribute to a better understanding of in vivo non-viral gene transfer using polymers, such as PEI and γ-PGA.

The role of fibronectin in pulmonary gene transfer following intravenous administration of lipoplex in mice.

We analyzed the effect of serum and fibronectin on pulmonary transgene expression after intravenous injection of cationic liposome-plasmid DNA (pDNA) complex (lipoplex) in mice. 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) methyl sulfate salt/cholesterol lipoplex was incubated with several serum components for 5 min at 37°C prior to injection. We analyzed pulmonary transgene expression and pulmonary accumulation of lipoplex. While interaction with serum did not decrease pulmonary transgene expression, interaction with heat-inactivated serum did decrease it. Moreover, interaction with fibronectin enhanced pulmonary transgene expression. Inhibition of the binding of fibronectin to integrin decreased pulmonary transgene expression after injection of untreated lipoplex. We found that pulmonary accumulation of lipoplex changed depending on the kind of interacting serum components after injection. Furthermore, interaction with fibronectin increased pulmonary accumulation of lipoplex. Interaction with serum was required for pulmonary gene transfer following intravenous injection of lipoplex. Fibronectin appears to be a particularly critical component. Furthermore, the binding of fibronectin interacting with lipoplex to integrin was an important mechanism for pulmonary transgene expression.

Diffusion coefficient of cationic liposomes during lipoplex formation determines transfection efficiency in HepG2 cells.

Cationic lipid-based lipoplexes are well-known for gene delivery. To determine the relationship between physicochemical characteristics and transfection efficiency, cationic liposomes of different sizes were prepared and incubated with plasmid DNA at different temperatures to form lipoplexes. We found that the liposome diffusion coefficient during lipoplex formation strongly correlated with the physicochemical characteristics of lipoplexes, accessibility of plasmid DNA in lipoplexes, and logarithm of gene expression per metabolic activity. Clathrin-mediated endocytosis was the major route for lipoplexes comprising 100 nm-liposomes, as reported previously. As liposome size increased, the major route shifted to lipid raft-mediated endocytosis. In addition, macropinocytosis was observed for all liposome sizes. The role of reactive oxygen species might depend on liposome size and endocytosis. Information from this study would be useful for understanding cationic lipoplex-mediated transfection.

Letrozole ameliorates liver fibrosis through the inhibition of the CTGF pathway and 17ß-hydroxysteroid dehydrogenase 13 expression.

BACKGROUND: To establish a treatment option for liver fibrosis, the possibility of the drug repurposing theory was investigated, with a focus on the off-target effects of active pharmaceutical ingredients. METHODS: First, several active pharmaceutical ingredients were screened for their effects on the gene expression in the hepatocytes using chimeric mice with humanized hepatocytes. As per the gene expression-based screening assay for 36 medications, we assessed the mechanism of the antifibrotic effect of letrozole, a third-generation aromatase inhibitor, in mouse models of liver fibrosis induced by carbon tetrachloride (CCl4) and a methionine choline-deficient (MCD) diet. We assessed liver histology, serum biochemical markers, and fibrosis-related gene and protein expressions in the hepatocytes. RESULTS: A gene expression-based screening assay revealed that letrozole had a modifying effect on fibrosis-related gene expression in the hepatocytes, including YAP, CTGF, TGF-ß, and CYP26A1. Letrozole was administered to mouse models of CCl4- and MCD-induced liver fibrosis and it ameliorated the liver fibrosis. The mechanisms involved the inhibition of the Yap-Ctgf profibrotic pathway following a decrease in retinoic acid levels in the hepatocytes caused by suppression of the hepatic retinol dehydrogenase, Hsd17b13 and activation of the retinoic acid hydrogenase, Cyp26a1. CONCLUSIONS: Letrozole slowed the progression of liver fibrosis by inhibiting the Yap-Ctgf pathway. The mechanisms involved the modification of the Hsd17b13 and Cyp26a1 expressions led to the suppression of retinoic acid in the hepatocytes, which contributed to the activation of Yap-Ctgf pathway. Because of its off-target effect, letrozole could be repurposed for the treatment of liver fibrosis. The third-generation aromatase inhibitor letrozole ameliorated liver fibrosis by suppressing the Yap-Ctgf pathway by partially modifying the Hsd17b13 and Cyp26a1 expressions, which reduced the retinoic acid level in the hepatocytes. The gene expression analysis using chimeric mice with humanized liver revealed that the mechanisms are letrozole specific and, therefore, may be repurposed for the treatment of liver fibrosis.

Efficient in vivo gene transfer by intraperitoneal injection of plasmid DNA and calcium carbonate microflowers in mice.

Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for in vivo experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers.

Pretreatment with epidermal growth factor enhances naked plasmid DNA transfer onto gastric serosal surface in mice.

We have developed a simple administration method, which is gastric serosal surface instillation of naked plasmid DNA (pDNA) in experimental animals. The purpose of this study was to improve gastric gene transfer efficiency by pre-treatment with a macropinocytosis enhancer, such as fetuin or epidermal growth factor (EGF), in mice. A series of concentrations of fetuin were instilled onto gastric serosal surface prior to instillation of naked pDNA in mice; however, fetuin did not improve transgene expression in the stomach 6 h after administration of pDNA. EGF also did not affect transgene expression in the stomach when pDNA was instilled immediately after EGF instillation. On the other hand, when pDNA was instilled onto gastric serosal surface 24 h after EGF treatment, transgene expression in the stomach was significantly improved by 2.6-fold. In addition, transgene-positive cells were increased 5.3-fold by EGF pre-treatment. High transgene expression in the stomach lasted for 48 h in the EGF pre-treatment group in comparison with that in the no pre-treatment group. These findings are valuable to develop an effective method of in vivo gene transfer to the stomach.

Flavonoids Enhance Lipofection Efficiency and Ameliorate Cytotoxicity in Colon26 and HepG2 Cells via Oxidative Stress Regulation.

The generation of reactive oxygen species (ROS) can affect cationic liposome-mediated transfection. In this study, we focused on a specific class of antioxidants, flavonoids, to investigate the transfection efficiency using cationic liposome/plasmid DNA complexes (lipoplexes) in 2D and 3D cultures of Colon26 and HepG2 cells, respectively. All tested flavonoids enhanced the transfection efficiency in 2D Colon26 and HepG2 cells. Among the tested flavonoids, 25 µM quercetin showed the highest promotion effect of 8.4- and 7.6-folds in 2D Colon26 and HepG2 cells, respectively. Transfection was also performed in 3D cultures of Colon26 and HepG2 cells using lipoplexes with quercetin. Quercetin (12.5 µM) showed the highest transfection efficiency at all transfection timings in 3D Colon26 and HepG2 cells with increased cell viability. Flow cytometry revealed that quercetin treatment reduced the population of gene expression-negative cells with high ROS levels and increased the number of gene expression-positive cells with low ROS levels in HepG2 cells. Information from this study can be valuable to develop strategies to promote transfection efficiency and attenuate cytotoxicity using lipoplexes.

Multiple components in serum contribute to hepatic transgene expression by lipoplex in mice.

BACKGROUND: Interaction of cationic liposome/plasmid DNA complex (lipoplex) with serum was not a limiting factor for in vivo transfection. After intraportal injection of lipoplex, hepatic transgene expression was enhanced by interaction with serum in mice. In the present study, we analyzed the mechanism of enhanced hepatic transgene expression of lipoplex by interaction with serum components. METHODS: Lipoplexes were incubated with several serum components for 5 min at 37 ° C before administration. Transfection efficiency of lipoplexes was measured 6 h after intraportal injection of lipoplex in mice. RESULTS: Depletion of divalent cation from serum decreased hepatic transgene expression. The addition of calcium ion to divalent cation-depleted serum restored transgene expression. Heat-inactivated serum and bovine serum albumin diminished the enhancing effect of serum on hepatic transgene expression. On the other hand, removal of anionic proteins from serum using an anion-exchanging column was critical for the enhancing effect of serum on transgene expression. Among the serum components tested, fibronectin and complement component C3 enhanced hepatic transgene expression. CONCLUSIONS: Hepatic transgene expression by lipoplex was enhanced by interaction with multiple components in serum. Interaction of lipoplex with serum could be an important factor for successful in vivo gene transfer. Hence, the information obtained in the present study is valuable for the future development of effective gene carriers.

Rubbing gastric serosal surface enhances naked plasmid DNA transfer in rats and mice.

We have developed in vivo gene transfer to mesothelial cells on the peritoneal organs, including the stomach. Simple instillation of naked plasmid DNA onto the gastric serosal surface in mice resulted in effective but transient transgene expression. Here, we developed a simple method to improve not only the transfection efficiency but also the duration of transgene expression. Rubbing the gastric serosal surface using a medical spoon immediately after instillation of naked plasmid DNA onto the gastric serosal surface resulted in 59-fold higher transgene expression 24 h after administration in rats. Without rubbing, transgene expression decreased under the detection limit 7 d after administration. On the other hand, rubbing the gastric serosal surface with a medical spoon after instillation of plasmid DNA prolonged transgene expression for one month. Mechanistic study in mice revealed that improved transfection should not be due to stimulation of cell function such as macropinocytosis by rubbing because rubbing before instillation of plasmid DNA did not improve transfection. Plasmid DNA should enter effectively into cells during rubbing. These findings are valuable to develop an effective method of in vivo gene transfer into peritoneal organs.