Stomach-selective gene transfer following the administration of naked plasmid DNA onto the gastric serosal surface in mice

The purpose of the present study was to achieve a stomach-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the gastric serosal surface in mice. Gene expression in the stomach and other tissues was evaluated by firefly luciferase activity. Six hours after gastric serosal surface instillation of naked pDNA, high gene expression in the stomach was observed. On the contrary, intravenous and intraperitoneal injection of naked pDNA exhibited no detectable gene expression. Following instillation of naked pDNA onto the gastric serosal surface, gene expression in the stomach was significantly higher than in other tissues. Gene expression in the stomach was highest 12 h after the instillation and thereafter decreased gradually. Utilizing a glass-made diffusion cell that is able to limit the contact dimension between the gastric serosal surface and the naked pDNA solution administered, site-specific gene expression in the stomach was achieved. This novel gene transfer method is expected to be a safe and effective treatment against serious stomach diseases.     

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.     

Effect of instillation method on the absorption of phenolsulphonphthalein as a model drug from the liver and small intestinal serosal surface in rats.

We have examined the effect of the instillation method on the absorption of a drug from the liver and the small intestinal serosal surface in rats. We performed continuous microinstillation via an infusion pump and bolus instillation via a syringe, using phenolsulphonphthalein (phenol red) as the model drug. After continuous microinstillation of phenolsulphonphthalein 2.35 mg in 235 microL for 5 min on the liver and small intestinal serosal surface in rats, the AUC (area under the curve) of the plasma concentration profile up to 60 min was significantly higher compared with bolus instillation. A similar trend was observed after continuous microinstillation of phenolsulphonphthalein 2.35 mg in 117.5 microL for 2.5 min. The calculated absorption rate constants (Ka) after continuous microinstillation of phenolsulphonphthalein based on a two-compartment model with first-order absorption were higher than those after bolus instillation on the liver and small intestinal serosal surface at either instillation concentration. Moreover, Ka was increased after continuous microinstillation of 2.35 mg in 117.5 microL at either instillation site. Instillation of phenolsulphonphthalein on the liver surface resulted in a 1.2- to 2.3-fold higher Ka compared with the small intestinal serosal surface. This tendency was marked after continuous microinstillation of 2.35 mg in 117.5 microL. In conclusion, absorption could be enhanced by instilling a small amount of drug solution on the liver surface gradually and continuously, suggesting a promising approach for instillation site-selective drug delivery in the peritoneal cavity.     

Stomach- and site-selective delivery of 5-fluorouracil following its application on the gastric serosal surface in rats

The present study was undertaken to elucidate the stomach- and site-selective delivery of 5-fluorouracil (5-FU) following its application on the gastric serosal surface in rats. An experimental system utilizing a cylindrical diffusion cell attached to the gastric serosal surface was established. To evaluate the gastric distribution of 5-FU, the stomach was separated into the site under the diffusion cell (site 1) and the site not under the diffusion cell (site 2). Furthermore, the mucosal side at site 1 was separated from the serosal side. After intravenous and oral administration of 5-FU, the 5-FU concentrations at sites 1 and 2 until 240 min were similar. After gastric serosal surface application of 5-FU, however, the concentration of 5-FU at site 1 until 240 min was approximately 10-fold higher than that at site 2, and was sustained. Furthermore, the 5-FU concentration on the mucosal side at site 1 and the serosal side at site 1 were comparable after gastric serosal surface application. The blood concentration of 5-FU was low (<4.4 microg/ml) until 240 min after gastric serosal surface application. The maximum blood concentration of 5-FU after gastric serosal surface application was significantly lower than after intravenous administration. Thus, the stomach- and site-selective delivery system following application on the gastric serosal surface could be applied with anticancer drugs for the treatment of gastric cancer.     

Spleen-selective gene transfer following the administration of naked plasmid DNA onto the spleen surface in mice

The purpose of present study was to examine spleen-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the spleen surface in mice. Gene expression in the spleen and other tissues was evaluated based on firefly luciferase activity. Six hours after spleen surface instillation of naked pDNA, high gene expression in the spleen was observed. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the spleen surface, gene expression in the spleen was significantly higher than those in other tissues. Six hours after instillation of naked pDNA onto the spleen surface, gene expression in the spleen reached the peak value, and thereafter decreased gradually. By utilizing a glass-made diffusion cell that is able to limit the contact dimension between the spleen surface and naked pDNA solution administered, site-specific gene expression in the spleen was found. This novel gene transfer method is expected to be a safe and effective strategy for DNA vaccine against serious infectious diseases and cancers.     

Unilateral kidney-selective gene transfer following the administration of naked plasmid DNA to the kidney surface in mice

We developed a gene transfer following the administration of naked plasmid DNA (pDNA) to the kidney surface in mice, and found that the luciferase levels produced in the applied kidney were significantly higher than those produced in another kidney. In contrast, stable renal gene expression was not observed in the case of intraperitoneal or intravenous administration of pDNA. The level of gene expression after instillation of pDNA to the kidney surface reached maximum at 12 h and gradually diminished thereafter. The production of luciferase was saturated at 5 microg of pDNA, and was not affected by instillation volume. Furthermore, pDNA uptake from the kidney surface was proved by in situ experiments using a glass-made diffusion cell. We demonstrated a novel unilateral kidney-selective gene transfer following the administration of naked pDNA to the kidney surface in mice.     

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.     

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.     

In vivo gene transfection via intravitreal injection of cationic liposome/plasmid DNA complexes in rabbits

To optimize the in vivo ocular transfection efficiency of plasmid DNA (pDNA)/cationic liposome complexes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoylphosphatidylethanolamine (DOPE) (1:1 molar ratio) liposomes and DOTMA/cholesterol (Chol) (1:1 molar ratio) liposomes were prepared with varying amounts of pDNA. pDNA/cationic liposome complexes were intravitreally injected (100 microL) in rabbits, and luciferase activity in the cornea, aqueous humor, iris-ciliary body, lens, vitreous body, and retina was measured. Transfection efficiency of pDNA alone did not change with pDNA ranging from 40 to 85 mg. In contrast, transfection efficiency of pDNA complexed with DOTMA/Chol liposomes significantly increased with the amount of pDNA ranging from 40 to 85 microg (P < 0.05). pDNA complexed with DOTMA/DOPE liposomes could not be prepared with pDNA greater than 60 microg. Among these experiments, pDNA (85 microg) complexed with DOTMA/Chol liposomes (pDNA:cationic liposome charge ratio (- : +) = 1.0:2.0) showed the highest transfection efficiency in the ocular tissue and its transfection-mediated luciferase activity peaked at 3 days. Among the ocular tissues, the highest gene expression was observed in the aqueous humor.     

Biodegradable nanoparticles composed of dendrigraft poly-l-lysine for gene delivery

We developed novel gene vectors composed of dendrigraft poly-l-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-l-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, γ-polyglutamic acid (γ-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/γ-PGA complexes) were shown to be stable nanoparticles, and those with γ-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/γ-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/γ-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/γ-PGA complexes for clinical gene therapy.     

Tissue-Specific Characteristics of <Emphasis Type="Italic">in Vivo</Emphasis> Electric Gene: Transfer by Tissue and Intravenous Injection of Plasmid DNA

Purpose To evaluate the tissue-specific characteristics of electric gene transfer after tissue and intravenous injection of naked plasmid DNA (pDNA).Methods pDNA encoding firefly luciferase was injected directly into the liver, kidney, spleen, skin and muscle, or into the tail vein of mice, and electric pulses were then applied to one of these organs. The distribution of transgene expressing cells was evaluated using pDNA encoding -galactosidase.Results Tissue injection of pDNA produced a significant degree of transgene expression in any tissue with the greatest amount in the liver, followed by kidney and spleen. The expression in these organs decreased quickly with time, and muscle showed the greatest expression at 7 days. Electroporation significantly increased the expression, and the expression level was comparable among the organs. Intravenous injection of pDNA followed by electroporation resulted in a significant expression in the liver, spleen, and kidney but not in the skin or muscle.Conclusions Electric gene transfer to the liver, kidney, and spleen can be an effective approach to obtain significant amounts of transgene expression by either tissue or intravenous injection of pDNA, whereas it is only effective after tissue injection as far as skin- or muscle-targeted gene transfer is concerned.     

Gene delivery system of pDNA using the blood glycoprotein fetuin

Fetuin is a biocompatible plasma protein and strongly enhances phagocytosis of bacteria, DNA and apoptotic cells by peripheral blood cells such as monocytes, macrophages and dendritic cells. We developed a novel gene delivery system: ternary complexes constructed with pDNA, polyethylenimine (PEI) and fetuin. Without covalent binding, fetuin was able to coat pDNA–PEI complexes, and stable anionic nanoparticles formed at a weight ratio greater than 30. Optimised pDNA–PEI–fetuin complexes significantly decreased the cytotoxicity of pDNA–PEI complexes in the melanoma cell line B16F10. Furthermore, the pDNA–PEI–fetuin complexes had higher transgene efficiency compared to that of commercial lipofectin previously reported in B16F10 cells despite an anionic surface. The pDNA–PEI–fetuin complexes did not agglutinate with erythrocytes. The pDNA–PEI–fetuin complexes had high gene expression in the spleen after intravenous administration in mice. Thus, the pDNA–PEI–fetuin complexes were a useful in vivo gene delivery system with tropism for the spleen.     

Liver site-specific gene transfer following the administration of naked plasmid DNA to the liver surface in mice

The present study was undertaken to investigate liver site-specific gene transfer following the administration of naked plasmid DNA (pDNA) to the liver surface in mice. We examined whether genes could be delivered to the liver site specifically by utilizing the glass-made diffusion cell that is able to limit the contact dimension between the liver surface and pDNA solution administered. Gene expression was detected at the site of diffusion cell attachment (site 1) and was significantly higher than in other liver sites and tissues. Moreover, gene expression was also detected at deeper site from the liver surface (noncontact side with pDNA solution). The level of gene expression at site 1 did not change significantly with pDNA treatment for 10, 30, and 60 min. In conclusion, we demonstrated that naked pDNA administered to the liver surface in mice was taken up from its surface, and subsequently the protein encoded by pDNA could be produced site specifically.     

Unilateral lung-selective gene transfer following the administration of naked plasmid DNA onto the pulmonary pleural surface in mice

The purpose of the present study was to examine unilateral lung-selective gene transfer following the administration of naked plasmid DNA (pDNA) onto the pulmonary pleural surface in mice. Naked pDNA was administered intravenously, intraperitoneally, and instilled onto the right pulmonary pleural surface. Four hours later, right pulmonary pleural surface instillation of naked pDNA resulted in high gene expression in the right lung. On the contrary, intravenous and intraperitoneal administration of naked pDNA resulted in no detectable gene expression. After instilling naked pDNA onto the right or left pulmonary pleural surface, gene expressions in the applied lung were significantly higher than those in the other lung and tissues. In addition, gene expressions were detected only in the intrathoracic tissues, not in the intraperitoneal tissues. Four hours after instillation of naked pDNA onto the right pulmonary pleural surface, gene expression in the right lung was the highest, and thereafter gene expression in the right lung decreased gradually. This novel gene transfer method is expected to be a safe and effective treatment against serious lung diseases.     

Effective intraperitoneal gene transfection system using nanobubbles and ultrasound irradiation

In this study, we demonstrate the low toxicity and highly efficient and spatially improved transfection of plasmid DNA (pDNA) with liposomal nanobubbles (bubble liposomes [BLs]) using ultrasound (US) irradiation in mice. Naked pDNA with BLs was intraperitoneally injected, followed by US irradiation. The injection volume, the duration of US irradiation, and the dose of BLs were optimized. Both BLs and US irradiation were essential to achieve high transgene expression from naked pDNA. We observed transgene expression in the entire peritoneal tissues, including the peritoneal wall, liver, spleen, stomach and small and large intestines. The area of transfection could be controlled with focused US irradiation. There were few changes in the morphology of the peritoneum, the peritoneal function or serum alanine aminotransferase levels, suggesting the safety of BLs with US irradiation. Using a tissue-clearing method, the spatial distribution of transgene expression was evaluated. BLs with US irradiation delivered pDNA to the submesothelial layer in the peritoneal wall, whereas transgene expression was restricted to the surface layer in the liver and stomach. Therefore, BLs with US irradiation could be an effective and safe method of gene transfection to the peritoneum.     

Effect of cationic charge on receptor-mediated transfection using mannosylated cationic liposome/plasmid DNA complexes following the intravenous administration in mice

The purpose of this study was to evaluate the effect of cationic charge of complexes after intravenous administration of cholesten-5-yloxy-N-[4-[(1-imino-2-D-thiomannosyl-ethyl)amino]butyl]formamide (Man-C4-Chol) containing cationic liposomes/pDNA complexes in mice. Transfection efficiency after intravenous administration of complex at a charge ratio (- : +) of 1.0:2.3 and/or 1.0:3.1 in liver and spleen expressing a mannose receptor on the cell surface were higher than those in lung. When complexes were formed at a charge ratio (- : +) of 1.0:4.7, on the other hand, transfection efficiency in the lung was highest, suggesting a non-specific interaction. Although asialoglycoprotein receptors are expressed on hepatocytes, a liver-selective gene transfection was not achieved by the intravenous administration of pDNA complexed with cholesten-5-yloxy-N-[4-[(1-imino-2-D-thiogalactosyl-ethyl)-amino]butyl]formamide (Gal-C4-Chol)/DOPE liposomes at a charge ratio (- : +) of 1.0 : 2.3. This information supports the design of pDNA/ligands-grafted cationic liposome complexes for cell-specific gene delivery after intravenous administration.     

Poly(l-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 ～ 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Chitosan microparticles containing plasmid DNA as potential oral gene delivery system.

The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. Chitosan has been shown to effectively bind DNA in saline or acetic acid solution and protect DNA from nuclease degradation. In this study, pDNA (plasmid DNA) was encapsulated in chitosan microparticles. Chitosan-DNA microparticles were prepared using a complex coacervation process and stability of plasmid DNA was investigated in this complex. The chitosan-DNA microparticles could protect the encapsulated plasmid DNA from nuclease degradation. Release of pDNA from microparticles was studied in simulated gastric, simulated intestinal medium and acidic PBS (phosphate buffer saline) (pH 4.5) buffer at 37 degrees C, and released pDNA was assayed spectrophotometrically. In vitro release of pDNA from chitosan microparticles was dependent on pH, as the pH of the release medium increased release profile decreased. In in vivo-animal studies blue color was observed with X-gal (4-chloro-5-bromo-3-indolyl-beta-galactosidase) staining of histological stomach and small intestine sections after oral administration of pDNA-chitosan microparticles as an indicator of exogeneous gene expression.     

Partial hepatectomy enhances polyethylenimine-mediated plasmid DNA delivery

Polyethylenimine (PEI) is widely used for non-viral transfection in vitro and in vivo. Hepatectomy is an interesting and considerable factor modifying PEI-mediated gene expression. We investigated the gene expression in mice over time following partial hepatectomy after an intravenous injection of PEI/plasmid DNA (pDNA) complex. pDNA encoding firefly luciferase was used as the model reporter gene. The hepatectomized liver was rapidly regenerated until 72 h. After 168 h, the liver weight of hepatectomized mice was similar to that of control mice. Slight liver function impairment was only observed at 1-24 h after hepatectomy in alanine aminotransferase and aspartate aminotransferase levels. Luciferase activity in the liver of partial hepatectomized mice at 48 h after partial hepatectomy increased by 70 times compared with that of control mice; however, luciferase activities did not significantly differ between hepatectomized mice and control mice in the spleen, lung, kidney, and heart. Among the lobes, luciferase activity by gram of tissue was not significantly different, indicating that gene expression enhancement by partial hepatectomy occurred equally throughout the liver. In conclusion, our findings demonstrate that liver resection is an influencing factor on PEI-mediated gene delivery in mice. These results indicate the necessity of considering cell division in PEI-mediated pDNA delivery.     

Influence of murine hepatitis induced by D-(+)-galactosamine hydrochloride and lipopolysaccharide on gene expression of polyethylenimine/plasmid DNA polyplex

We investigated the influence of murine hepatitis induced by D-(+)-galactosamine and lipopolysaccharide (D-GalN/LPS) on polyethylenimine (PEI)-mediated plasmid DNA (pDNA) delivery. pDNA encoding firefly luciferase was used as the model reporter gene. PEI was used as the non-viral vector because of its high gene expression and low toxicity. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice indicated the highest peaks at 12 h after D-GalN/LPS injection, then the activities of serum ALT and AST rapidly decreased. We determined luciferase activity in various organs of D-GalN/LPS-treated mice and control mice after an intravenous administration of PEI/pDNA complexes. High transgene expression was observed in the liver, spleen, and lung of both mice. Compared to the control mice, a significant increase of transgene expression was observed in the liver of D-GalN/LPS-treated mice after D-GalN/LPS injection. The transgene expression in the spleen and lung decreased at 6 and 12 h after D-GalN/LPS injection. In conclusion, we found that murine hepatitis induced by D-GalN/LPS injection can influence PEI-mediated pDNA delivery and its influence was different from that induced by CCl(4) injection which was reported previously. These results demonstrated the necessity of considering the timing and dose of gene therapy according to the disease and its stage.