Preliminary study to determine the optimal conditions for the simultaneous complexation of siRNA and plasmid DNA

We investigated the complexation ratio of siRNA and plasmid DNA combined with Lipofectamine for the effective delivery of genetic materials. First, the amount of Lipofectamine was varied with a fixed amount of DNA or siRNA to determine in which proportions they would form an optimal combination. Finally, to investigate the effect of DNA or siRNA on the co-complexation of both DNA and siRNA, the co-complex of DNA and siRNA was prepared at the various ratios with a fixed amount of DNA. All complexation was confirmed by gel retardation of DNA or siRNA on agarose gels. The effects of siRNA complexes on mRNA expression from plasmid DNA were explored post-transfection, while the influence of plasmid DNA complexes on the transfection of siRNA was determined in GFP-expressing H4IIE cells. The complex between DNA and Lipofectamine was formed at a weight ratio of 0.8:1, whereas the light band of siRNA/Lipofectamine disappeared at a weight ratio of 4:1. When the amounts of DNA, siRNA, and the mixture were fixed, the optimal ratio of nucleic acids and Lipofectamine in our composition was 110:80:350 (ng). Confocal images and flow cytometry showed that inhibition of GFP expression by siRNA was not interfered with by co-complexed plasmid DNA. Moreover, mRNA expression of adiponectin was not hampered by the addition of siRNA; rather, it was increased. Thus, co-complexation of siRNA and plasmid DNA may have a synergistic effect on delivery of the therapeutic gene and siRNA.     

Prenatal diagnosis of coarctation of the aorta with ventricular septal defect: A case report

Objective

To present an accurate prenatal diagnosis of coarctation of the aorta with ventricular septal defect and to illustrate how early diagnosis in prenatal period with proper referral and counseling can optimize management.

Encapsulation enhancement and stabilization of insulin in cationic liposomes

The purpose of this study was to enhance encapsulation efficiency and sustained-release delivery for parenteral administration of a protein drug. To reduce the administration frequency of protein drugs, it is necessary to develop sustained delivery systems. In this study, protein drug-loaded cationic liposomes were formulated with dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE/DOTAP/CH of 2/1.5/2. Five mol% of distearoylphosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) was added prior to encapsulation of the drug into liposomes. Insulin was chosen as a model protein drug and encapsulation efficiency was evaluated in various liposomes with and without DSPE-PEG. Scanning electron microscopy was used to examine the insulin-loaded cationic liposomes. Structural analysis was performed using spectropolarimetry. Additionally, the stability and cytotoxicity of insulin-loaded cationic liposomes were evaluated. Liposomes coated with DSPE-PEG showed higher insulin encapsulation efficiency than did those without DSPE-PEG, but not significantly. Moreover, among the liposomes coated with DSPE-PEG, those hydrated with 10% sucrose showed higher encapsulation efficiency than did liposomes hydrated in either phosphate-buffered saline or 5% dextrose. In vitro release of insulin was prolonged by cationic liposomes. Our findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of protein and peptide drugs to prolong efficacy and improve bioavailability.     

Changes in caregiver burden among informal caregivers of stroke patients in Mongolia

Background: Modern therapeutics and health care improvements prolong stroke patients’ survival; however, the degree of disability remains high. Stroke survivors often require caregivers, particularly in the first year after the onset of the stroke. Longitudinal assessment of and factors associated with caregiver burden (CGB) among caregivers of stroke patients has been scarcely discussed. This study aimed to define the changes in CGB in the first year of caregiving among the caregivers of stroke patients and to identify associated factors.

Methods: A prospective, multi-centered observational study was conducted in nine public hospitals in Mongolia. We used the Montgomery CGB Scale for assessing CGB, and repeated the assessment after 1 year. Stroke patient characteristics were included in the analyses. Multinomial logistic regressions were conducted to analyze changes in CGB.

Results: A paired t-test analysis revealed that demand burden increased (from 12.61 to 11.50, p = 0.034), whereas stress burden decreased (from 10.69 to 11.60, p = 0.016). Although objective burden decreased, the difference was not significant. Factors associated with these changes in CGB were the caregiver’s marital status, the caregiver’s relationship with the patient, financial difficulties, and the patient’s sex and dependency.

Discussion: The information on factors predicting changes in CGB in the first year of caregiving provided in this study suggests that social or financial support can assist in reducing CGB among the caregivers of post-stroke patients.     

17-beta estradiol inhibits oxidative stress-induced accumulation of AIF into nucleolus and PARP1-dependent cell death via estrogen receptor alpha

Oxidative stress-induced DNA damage results in over-activation of poly(ADP-ribose) polymerase 1 (PARP1), leading to parthanatos, a newly discovered cell elimination pathway. Inhibition of PARP1-dependent cell death has shown to improve the outcome of diseases, including stroke, heart ischemia, and neurodegenerative diseases. In the present study we aimed to detect whether estrogen plays a protective role in inhibiting parthanatos. We utilized human mammary adenocarcinoma cells (MCF7) that abundantly express the estrogen receptor alpha and beta (ERα and ERβ). Parthanatos was induced by challenging the cells with hydrogen peroxide (H₂O₂). Microscopic imaging and molecular biological techniques, such as Western blot analysis and RNA interference, were performed. The results showed 17β estradiol (E2) protected MCF7 cells from PARP1-dependent cell death by decreasing protein PARylation, and AIF translocation into nuclei/nucleoli. Down-regulation of ERα expression by siRNA before E2 addition resulted in the failure of the E2-mediated inhibition of H₂O₂-induced protein PARylation and AIF nucleolar translocation. Together these data suggest that estrogen via its alpha-type receptor inhibits oxidative stress-induced, PARP1-dependent cell death. The present study provided us insight into how to apply hormone therapy in intervention of parthanatos-implicated ischemic and degenerative diseases.     

Formulation parameters influencing the physicochemical characteristics of rosiglitazone-loaded cationic lipid emulsion

To enhance the solubility of rosiglitazone, rosiglitazone-loaded cationic lipid emulsion was formulated using cationic lipid DOTAP, DOPE, castor oil, tween 20, and tween 80. The formulation parameters in terms of droplet size were optimized focused on the effect of the cationic lipid emulsion composition ratio on drug encapsulating efficiency, in vitro drug release, and cellular uptake of the rosiglitazone-loaded emulsion. Droplet sizes of a blank cationic emulsion and a rosiglitazone-loaded cationic emulsion ranged between 195-230 nm and 210-290 nm, respectively. The encapsulation efficiency of the rosiglitazone-loaded emulsion was more than 90%. The rosiglitazone-loaded cationic emulsion improved in vitro drug release over the drug alone and showed a much higher cellular uptake than rosiglitazone alone. Moreover, drug loading in cationic emulsions increased cellular uptake of rosiglitazone in insulin-resistant HepG2 cells more than the normal HepG2 cells. Taken together, these results indicate that cationic lipid emulsions could be a potential delivery system for rosiglitazone and could enhance its cellular uptake efficiency into target cells.