Yuhuan Li

Development of liposomal ginsenoside Rg3: Formulation optimization and evaluation of its anticancer effects

The Ginsenoside Rg3 has been shown to possess antiangiogenic and anticancer properties. Because of its limited water solubility, we decided to design and synthesize liposomal Rg3 (L-Rg3), to optimize preparation conditions, and to investigate further whether liposome could enhance the anticancer activity of Rg3. L-Rg3 was prepared using a film-dispersion method and the preparation conditions were optimized with response surface methodology (RSM). The mean encapsulation efficiency (EE) of 82.47% was close to the predicted value of 89.69%. Therefore, the optimized preparation condition was predicted correctly. We evaluated the cytotoxicity, pharmacokinetics, biodistribution and antitumor activities of L-Rg3. HepG2 and A549 cells were treated with Rg3 or L-Rg3 at different concentrations in vitro. Pharmacokinetics and biodistribution studies were carried out in Wistar rats. Tumor model was established by inoculating a suspension of A549 cells into BALB/c nude mice. The mice were divided into Saline, Rg3 solution, and L-Rg3 groups with the drug given by i.p. injection. Survival of the mice and tumor volume were monitored. In addition, CD34 immunohistochemical analysis was used for measuring microvessel density (MVD) of the tumor tissues. The cytotoxicity and ratio of tumor inhibition of L-Rg3 group were significantly higher than the Rg3 solution group. MVD values in the Rg3 solution and L-Rg3 groups decreased, especially in the L-Rg3 group. Compared to Rg3 solution, the L-Rg3 showed increased Cmax and AUC of Rg3 by 1.19- and 1.52-fold, respectively. This liposomal formulation could potentially produce a viable clinical agent for improving the anticancer activity of Rg3.

Silencing of Survivin Expression Leads to Reduced Proliferation and Cell Cycle Arrest in Cancer Cells.

Survivin is an anti-apoptotic gene that is overexpressed in most human tumors. RNA interference using short interfering RNA (siRNA) can be used to specifically inhibit survivin expression. Tumor cells were treated with a newly designed survivin siRNA, which was modified with 2′-OMe. Cellular survivin mRNA and protein levels were determined by real-time qRT-PCR and Western blot, respectively. Cell cycle and apoptosis were determined by flow cytometry. Cell proliferation was measured by MTT assay. Our data showed that the novel survivin-targeted siRNA could efficiently knockdown the expression of survivin and inhibit cell proliferation. Survivin mRNA was reduced by 95% after 48h treatment with 20nM siRNA. In addition, the siRNA could markedly arrest the cell cycle at the G2/M checkpoint and induce cellular apoptosis in a dose-dependent manner. The percentage of apoptotic cells reached 50% when treated with 40nM siRNA. In conclusion, we have identified a novel chemically modified siRNA against survivin that is highly efficient and delineated its mechanism of action, thus demonstrating a potential therapeutic role for this molecule in cancer. Further evaluation of this siRNA for therapeutic activity is warranted.

Fatty acid modified octa-arginine for delivery of siRNA

Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Four fatty acids derivatives of octa-arginine (R8) were synthesized and evaluated for the delivery of siRNA into hepatocellular carcinoma Hep G2 and human lung adenocarcinoma A549 cells. The results showed that the long chain acid oleic acid or stearic acid derivatives of R8, OA-R8 and StA-R8, were more efficient in siRNA complexation and form nanoparticles with greater stability compared to the native R8. Cellular uptake of fluorescence-labeled siRNA delivered by OA-R8 and StA-R8 in Hep G2 and A549 cells was substantially 40-50 times higher than unmodified R8. A significant reduction in siRNA cellular uptake was observed in the presence of sucrose and cytochalasin D, indicating endocytosis as a primary mechanism of cellular entry. A survivin siRNA was used to prepare nanoparticles with OA-R8 or StA-R8 and evaluated for silencing of survivin mRNA and protein in A549 cells, and the inhibition efficiencies of survivin protein reached to 50.3% and 54.6%, respectively. The results showed greater effectiveness with the derivatized R8. Taken together, these findings showed that long chain fatty acid derivatives of R8 are efficient delivery agents for siRNA and may facilitate its therapeutic application.

Single-Step Microfluidic Synthesis of Transferrin-Conjugated Lipid Nanoparticles for siRNA Delivery

Microfluidic systems can accelerate clinical translation of nanoparticles due to their ability to generate nanoparticles in a well-controlled and reproducible manner. In this study, a single-step process based on microfluidic focusing (MF) was employed to synthesize transferrin-conjugated lipid nanoparticles (Tf-LNPs) and the method was compared with a multi-steps bulk mixing (BM) method. The results indicate that this single-step MF process enables rapid and efficient synthesis of Tf-LNPs, which were named Tf-LNPs-MF. Tf-LNPs-MF was shown to have a smaller size and more uniform structures compared to LNPs produced by multi-steps BM method (Tf-LNPs-BM). Furthermore, efficient cellular uptake of Tf-LNPs-MF in vitro as well as greater tumor inhibition in vivo proved that Tf-LNPs-MF had higher siRNA delivery efficiency in vitro and in vivo. Taken together, this single-step microfluidic synthesis significantly simplified the Tf-LNPs production and improved their drug delivery properties and may serve as a valuable tool for developing new cancer therapies.

Antidiabetic and Antinephritic Activities of Aqueous Extract of Cordyceps militaris Fruit Body in Diet-Streptozotocin-Induced Diabetic Sprague Dawley Rats

Cordyceps militaris has long been used as a crude drug and folk tonic food in East Asia. The present study aims to evaluate the antidiabetic and antinephritic effects of the aqueous extract of the Cordyceps militaris fruit body (CM) in diet-streptozotocin- (STZ-) induced diabetic rats. During four weeks of continuous oral administration of CM at doses of 0.5, 1.0, and 2.0 g/kg and metformin at 100 mg/kg, the fasting blood glucose and bodyweight of each rat were monitored. Hypoglycemic effects of CM on diabetic rats were indicated by decreases in plasma glucose, food and water intake, and urine output. The hypolipidemic activity of CM was confirmed by the normalization of total cholesterol, triglycerides, and low- and high-density lipoprotein cholesterol in diabetic rats. Inhibitory effects on albuminuria, creatinine, urea nitrogen, and n-acetyl-β-d-glucosaminidase verified CMs renal protective activity in diabetic rats. Furthermore, CM exerted beneficial modulation of inflammatory factors and oxidative enzymes. Compared with untreated diabetic rats, CM decreased the expression of phosphor-AKT and phosphor-GSK-3β in the kidneys. Altogether, via attenuating oxidative stress, CM displayed antidiabetic and antinephritic activities in diet-STZ-induced diabetic rats.