Rubbing Gastric Serosal Surface Enhances Naked Plasmid DNA Transfer in Rats and Mice

Abstract

The stomach is a digestive organ that is essential for nutrient intake. Alcohol, drugs, stress, and Helicobacter pylori reduce barrier function against acidic pH, subsequently causing gastritis and gastric ulcer. Moreover, gastric cancer is one of the most common malignant tumors in the world. The mortality due to Japanese gastric cancer was over 50000 deaths annually according to the Center for Cancer Control and Information Services, National Cancer Center, Japan. Surgical resection of gastric cancer without metastasis is a first-line therapy and provides relatively good prognosis compared with that of other cancers. However, recurrence, liver metastasis, and/or peritoneal dissemination in gastric cancer are serious problems associated with high mortality. Advanced gastric cancer does not generally respond to conventional chemotherapy or radiotherapy. 1)Gene delivery to the stomach is a rational approach to treat gastric ulcer and cancer since various genes are correlated with these diseases.2) There are mainly three routes to deliver a transgene to the stomach: mucosal route, 3) direct injection, 4) and serosal route. 5-7) Gene transfer via the mucosal route is hardly possible owing to the barrier function of the mucosal layer of the stomach. Direct injection is effective to transfer a transgene; however, the area of transfection is restricted to the injection site(s). The serosal route, which we have developed, 5-7) is attractive for treatment against serosal invasion of gastric ulcer and cancer. Interestingly, simple instillation of naked plasmid DNA solution onto organ surfaces, including liver, 8) kidney, 9) spleen, 10) and gastric serosal surfaces, 5-7) resulted in effective transgene expression in mice and rats. We already elucidated that the mechanism of naked plasmid DNA transfer was uptake via Rac-mediated macropinocytosis.11) However, we were not sure whether the transfection efficiency was sufficient to treat gastric diseases. Moreover, the duration of transgene expression was relatively short. Thus, we considered it necessary to improve transgene expression.We hypothesized two strategies to improve transgene expression. One is enhancement of Rac signaling pathway controlling macropinocytosis of plasmid DNA. However, protein kinase C enhancer (phorbol 12-myristate 13-acetate, PMA), which is one of the macropinocytosis enhancers, did not affect transgene expression after naked plasmid DNA transfer onto the gastric serosal surface in mice (data not shown). Another strategy is utilization of physical force(s). With regard to physical forces, several methods have been reported, such as electroporation 12) and use of a gene gun. 13) However, both methods require specialized devices. Here, we developed a simple method to enhance transgene expression in gastric serosal surface cells by rubbing the surface using a medical spoon after instillation of naked plasmid DNA in rats and mice. This simple rubbing method improved not only transfection efficiency but also duration of transgene expression. MATERIALS A...

Pretreatment with Epidermal Growth Factor Enhances Naked Plasmid DNA Transfer onto Gastric Serosal Surface in Mice

“…43) 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 pDNA did not improve transfection. pDNA should enter effectively into cells during rubbing.…”

Combination of Nanoparticles with Physical Stimuli toward Cancer Therapy

“…42) On the other hand, rubbing stimuli against the gastric serosal surface greatly enhanced naked pDNA transfer. 43) In addition, the concomitant use of an abrasive compound calcium carbonate with naked pDNA was effective to a similar extent as the rubbing stimuli. 44) These physical stimuli would improve the efficacy of the nanoparticulate systems.…”

“…[55][56][57] As mentioned above, physical stimuli such as rubbing the stomach surface could enhance the transgene expression. 43) Thus, appropriate selection of the administration routes would also be an important issue in the physical stimuli-mediated delivery of the nanoparticles. 58) In general, endocytosis is the primary cellular uptake mechanism of nanoparticles.…”

Targeted Gene Delivery: Importance of Administration Routes

“…Pretreatment with epidermal growth factor (EGF) enhanced transgene expression and increased transgene-positive cells on the stomach after instillation of naked plasmid DNA onto it [162]. Rubbing the gastric serosal surface with a medical spoon after instillation of naked plasmid DNA onto the stomach was more effective than EGF pretreatment [163]. However, rubbing the organ surface with a medical spoon may be impractical for future clinical application.…”