Teruyoshi Aoyama

Liver targeting of plasmid DNA by pullulan conjugation based on metal coordination.

Liver targeting of plasmid DNA was achieved through conjugation of pullulan derivatives with chelate residues based on metal coordination. Triethylenetetramine (Ti), diethylenetriamine pentaacetic acid (DTPA), and spermine (Sm) were chemically introduced to pullulan, a polysaccharide with an inherent affinity for the liver, to obtain various pullulan-Ti, pullulan-DTPA, and pullulan-Sm derivatives. Irrespective of the type of pullulan derivatives, intravenous injection of the pullulan derivatives-plasmid DNA conjugates with Zn2+ coordination significantly enhanced the level of gene expression only in the liver to a significant greater extent than that of free plasmid DNA. The enhanced gene expression by the pullulan-DTPA-plasmid DNA conjugate was specific to the liver and the level was significantly higher than that of the pullulan-DTPA-plasmid DNA mixture. The level of gene expression depended on the percentage of chelate residue introduced, the mixing ratio of the plasmid DNA-DTPA residue in conjugate preparation, and the plasmid DNA dose. The gene expression induced by the conjugate lasted over 12 days after injection. A fluorescent-microscopic study revealed that the plasmid DNA was localized at the liver after injection of the pullulan-DTPA-plasmid DNA conjugate with Zn2+ coordination. Pre-injection of both arabinogalactan and galactosylated albumin suppressed significantly the liver level of gene expression, in contrast to that of mannosylated albumin, indicating that the plasmid DNA in the conjugate was transfected at hepatocytes. We conclude that the Zn2+-coordinated pullulan conjugation is a promising way to enable the plasmid DNA to target to the liver for gene expression as well as to prolong the time duration of gene expression

Local Delivery of Matrix Metalloproteinase Gene Prevents the Onset of Renal Sclerosis in Streptozotocin-Induced Diabetic Mice

The present study was undertaken to investigate whether matrix metalloproteinase (MMP) functions to prevent the occurrence of destructive fibrosis in progressive renal disease. As a sustained release carrier of plasmid DNA, biodegradable hydrogels and microspheres were formulated from cationized gelatin prepared through aminization. Plasmid DNA was released from the cationized gelatin hydrogels as a result of hydrogel degradation. A plasmid DNA including a cytomegalovirus promoter and human recombinant MMP-1 gene (pCMV-MMP) was constructed. Gelatin microspheres incorporating pCMV-MMP as well as phosphate-buffered saline (PBS) with or without pCMV-MMP were injected into the renal subcapsule of C57BL/6 mice, which were intraperitoneally injected with streptozotocin (STZ) to induce diabetes 7 days after operation. The mice were killed 4 weeks after STZ injection to sample their blood and kidneys for biochemical and histological examinations. An immunofluorescence study confirmed that MMP protein was expressed around the renal tissue injected with gelatin microspheres incorporating pCMV-MMP. When applied with cationized gelatin microspheres incorporating pCMV-MMP, the mice showed a level of blood urea nitrogen significantly lower than that of other groups. A reduced content of collagen in the kidneys of mice administered gelatin microspheres incorporating pCMV-MMP was histologically observed. Further, the hydroxyproline assay revealed a significantly decreased content of hydroxyproline in kidney. We conclude that sustained release of MMP-1 gene is a promising prophylactic trial for kidney fibrolysis and dysfunction in the STZ-induced diabetic mouse model.

Ultrasound Enhancement of In Vitro Transfection of Plasmid DNA by a Cationized Gelatin

In vitro transfection efficiency of a plasmid DNA for rat gastric mucosal (RGM)-1 cells was enhanced by ultrasound (US) irradiation. Ethylenediamine was introduced to the carboxyl groups of gelatin to prepare a cationized gelatin as the vector of plasmid DNA encoding luciferase. An electrophoresis experiment revealed that the cationized gelatin was mixed with plasmid DNA at the weight ratio of 5.0 to form a cationized gelatin-plasmid DNA complex. The complex obtained was about 200 nm in diameter with a positive charge. When incubated with the cationized gelatin-plasmid DNA complex and subsequently exposed to US, RGM-1 cells exhibited a significantly enhanced luciferase activity although the extent increased with an increase in the DNA concentration, in contrast to the cationized gelatin alone with or without US irradiation and US irradiation alone. US irradiation was also effective in enhancing the activity by free plasmid DNA although the extent was less than that of the complex. The US-induced enhancement of luciferase activity was influenced by the exposure time period, frequency, and intensity of US. The activity enhancement became higher to be significant at the irradiation time period of 60 s and thereafter decreased. A series of cytotoxicity experiments revealed that an increase in the irradiation time period and intensity of US decreased the viability of cells themselves. It is possible that US irradiation under an appropriate condition enables cells to accelerate the permeation of the cationized gelatin-plasmid DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. These findings clearly indicate that US exposure is a simple and promising method to enhance the gene expression of plasmid DNA.

Ultrasound Enhances the Transfection of Plasmid DNA by Non-viral Vectors

Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

Enhanced expression of plasmid DNA-cationized gelatin complex by ultrasound in murine muscle

This study is an investigation to experimentally confirm that ultrasound (US) irradiation is effective in enhancing the gene expression of plasmid DNA. A cationized gelatin was prepared by introducing primary amino groups into gelatin. The plasmid of the LacZ gene was complexed with the cationized gelatin and injected into the femoral muscle of normal mice. Following US irradiation at the injected site of muscle, the gene expression of the treated muscle was evaluated to compare with that of the complex injection plus no US irradiation. US irradiation enabled the complex to significantly enhance the gene expression at the injected muscle, in contrast to naked plasmid DNA, although the extent depended on the experimental conditions, such as the injection dose of plasmid DNA, and the time period or timing of US irradiation. The gene-expressed area at the muscle was not changed by the time interval between the complex injection and US irradiation. Fluorescent microscopic observation revealed that the complex was homogeneously distributed in the muscle tissue around the injected site by US irradiation without any dissociation of plasmid DNA. The fluorescent image of plasmid DNA superposed on that of myosin heavy chain indicated intracellular localization of plasmid DNA. We demonstrated that US irradiation is a promising technique to enhance gene expression even in the normal muscle.

In Vitro Transfection of Plasmid DNA by Amine Derivatives of Gelatin Accompanied with Ultrasound Irradiation

AbstractPurpose. The purpose of this study is to examine the ultrasound (US)-enhanced gene expression by the complexes of a plasmid DNA with gelatin derivatives of aminization. Methods. Gelatin derivatives with different introduced extents of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were prepared with a water-soluble carbodiimide. The molecular size and zeta potential of the gelatin derivatives before and after complexation with the plasmid DNA were examined. After incubation with the complexes with or without US exposure, the DNA expression of rat gastric mucosal cells was measured to evaluate the effect of the type of gelatin derivatives on their gene expression. The cell uptake of the complexes, the cell viability, and the buffering effect of gelatin derivatives were examined. Results. The apparent molecular size and zeta potential of gelatin derivatives became larger as their aminization extent increased although the Sm gelatin derivative of higher aminization showed a larger value than other corresponding derivatives. Irrespective of the type of gelatin derivatives, the apparent molecular size of plasmid DNA was reduced by increasing the gelatin-DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin-DNA complexes showed the zeta potential of 10-15 mV. The cells incubated with the complex exhibited significantly stronger luciferase activities than free plasmid DNA, and the activity was further enhanced by US irradiation. The enhancement was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. The amount of plasmid DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative, whereas US irradiation did not significantly increase the DNA internalization. US irradiation had no effect on the viability of cells incubated with every gelatin derivative-plasmid DNA complex, although the viability was decreased by the complex incubation. The buffering capacity of Sm derivative was higher than that of Ed and Sd derivatives and comparable with that of polyethylene amine. Conclusion. Among amine derivatives of gelatin, the Sm derivative enabled the plasmid DNA to induce the US-enhanced gene expression of cells in vitro most effectively because of the superior buffering effect.

RETRACTED: Tumor targeting of gene expression through metal-coordinated conjugation with dextran

Tumor targeting of plasmid DNA was achieved through the conjugation of dextran derivatives with chelate residues based on metal coordination. Diethylenetriamine pentaacetic acid (DTPA), spermidine (Sd), and spermine (Sm) were chemically introduced to the hydroxyl groups of dextran to obtain dextran-DTPA, dextran-Sd and dextran-Sm derivatives. Conjugation of the dextran derivative by Zn(2+) coordination decreased the apparent size of the plasmid DNA, depending on the derivative type. The negative zeta potential of plasmid DNA became almost 0 mV after Zn(2+)-coordinated conjugation with dextran-Sm. When the dextran derivative-plasmid DNA conjugates with Zn(2+) coordination were intravenously injected subcutaneously into mice bearing Meth-AR-1 fibrosarcoma, the dextran-Sm-plasmid DNA conjugate significantly enhanced the level of gene expression in the tumor, in contrast to the conjugate of other dextran derivatives and free plasmid DNA. The enhanced gene expression produced by the Zn(2+)-coordinated dextran-Sm-plasmid DNA conjugate was specific to the tumor, whereas a simple mixture of dextran-Sm and plasmid DNA was not effective. The level of gene expression depended on the percentage of chelate residues introduced, the mixing weight ratio of the plasmid DNA/Sm residue used for conjugate preparation, and the plasmid DNA dose. A fluorescent microscopic study revealed that localization of plasmid DNA in the tumor tissue was observed only after injection of the dextran-Sm-plasmid DNA conjugate with Zn(2+) coordination. In addition, the gene expression induced by the conjugate lasted for more than 10 days after the injection. We conclude that Zn(2+)-coordinated dextran-Sm conjugation is a promising way to enable plasmid DNA to target the tumor in gene expression as well as to prolong the duration of gene expression.

ROLE OF VOLUME WEIGHTED MEAN NUCLEAR VOLUME FOR PREDICTING DISEASE OUTCOME IN PATIENTS WITH RENAL CELL CARCINOMA

PURPOSE Histological grading of malignancy in renal cell carcinoma is based on qualitative, morphological examination and suffers from poor reproducibility. On the other hand, estimate of volume weighted mean nuclear volume, which was developed based on a stereological technique, is an easy method to perform with high reproducibility. Furthermore, it has been reported that mean nuclear volume is remarkably correlated with prognosis of bladder and prostate cancer. We compared mean nuclear volume to a histological grading method and TNM classification in determining the prognosis of renal cell carcinoma using a Cox proportional hazards model. MATERIALS AND METHODS A retrospective, prognostic study was done of 65 patients with renal cell carcinoma diagnosed by radical nephrectomy or needle punch biopsy between January 1978 and November 1995. Unbiased estimates of mean nuclear volume were compared to TNM classification and histological grading to determine prognostic value. RESULTS Univariate analysis indicated that all TNM classifications, histological grades and estimates of mean nuclear volume were significantly correlated with prognosis of renal cell carcinoma. However, multivariate analysis revealed that estimates of mean nuclear volume and metastasis at diagnosis were the 2 most powerful independent predictors of disease specific survival. CONCLUSIONS Our results indicate that estimates of mean nuclear volume are prognostically superior to morphological grading of renal cell carcinoma. We recommend mean nuclear volume estimates as an adjunct to subjective histological grading in patients with this disease.

Prognostic Criteria in Patients with Prostate Cancer: Correlation with Volume Weighted Mean Nuclear Volume

PURPOSE Grading of malignancy in prostate cancer is based on qualitative, morphological examination and suffers from poor reproducibility. On the other hand, estimating the volume weighted mean nuclear volume, which was developed by Gunderson and Jensen based on a stereological technique, is a simple method with high reproducibility. Furthermore, it has been reported that mean nuclear volume is remarkably correlated with prognosis of bladder cancer. We compared mean nuclear volume to 2 histological grading methods and clinical stage to determine the prognosis of prostate cancer using a Cox proportional hazards model. MATERIALS AND METHODS A retrospective, prognostic study of 52 patients with prostate cancer diagnosed by transurethral resection of the prostate or needle punch biopsy between January 1983 and July 1994 was performed. Unbiased estimates of mean nuclear volume were compared to patient age at diagnosis, clinical stage, histological grading according to the World Health Organization (WHO) classification and Gleason score of the prognostic value. RESULTS Univariate analysis revealed that estimates of mean nuclear volume (p = 0.0011), clinical stage (p = 0.0014) and WHO classification (p = 0.0010) correlated significantly with progression-free survival, and that clinical stage (p = 0.0005) and estimates of mean nuclear volume (p = 0.0049) correlated significantly with disease-specific survival of patients with prostate cancer. However, multivariate analysis revealed that only estimates of mean nuclear volume and clinical stage were the 2 most powerful independent prognosticators in progression-free and disease-specific survival. CONCLUSIONS Our results indicate that estimates of mean nuclear volume are prognostically superior to morphological grading of malignancy, such as Gleason score and WHO classification, in prostate cancer.

Bilateral Renal Cell Carcinoma in a Patient with Tuberous Sclerosis

A case of pure bilateral renal tell carcinoma (RCC) in a 21‐year‐old female diagnosed as having tuberous selerosis is reported. She underwent a left nephrectomv because of her loss of appetite possibly caused by the tumor compressing her intestines. The preoperalive CI scan showed the presence of adipose tissue in bilateral renal tumors, which is highly suggestive of angiomvolipoma (AML). Histological examination, however, revealed no area or component ot the tumor with features characteristic of AML.