A novel method for the development of plasmid DNA-loaded nanoliposomes for cancer gene therapy.

We aimed to develop a simple yet novel method to prepare plasmid DNA-loaded nanoliposomes for cancer gene therapy. Murine interleukin-12 (mIL-12) pDNA-loaded nanoliposomes were prepared via novel freeze-drying of a monophase solution method. The physicochemical characteristics, cytotoxicity, and transfection efficiency of the prepared nanoliposomes in murine CT-26 colon carcinoma cells were evaluated. Furthermore, tumor progression and survival rate in CT-26 colon carcinoma-bearing BALB/c mice subsequent to direct intratumoral injections were investigated over a period of 40 days. Using this preparation method, nanoliposomes with particle size of around 300 nm and zeta potential of 96.5 mV were obtained. The transmission electron microscope results showed that the liposomes were nano-sized and almost spherical. The agarose gel retardation assay revealed the pDNA encapsulation in the nanoliposomes. The nanoliposomes with 72.4% encapsulation efficiency and low cell toxicity could significantly improve mIL-12 expression by approximately 25-fold relative to the naked mIL-12 pDNA. There was a significant tumor growth inhibition after repeated injections of mIL-12 pDNA-loaded nanoliposomes. This is the first study on the freeze-drying of a monophase solution method as a simple yet novel technique for the preparation of pDNA-loaded nanoliposomes. Given the ease of preparation method and promising in vitro and in vivo characteristics, this investigation demonstrates advances in pDNA lipid formulation for cancer gene therapy.

Apoptotic Effect of Saccharomyces cerevisiae on Human Colon Cancer SW480 Cells by Regulation of Akt/NF-ĸB Signaling Pathway.

Drug resistance is one of the major problems, which causes recurrence of cancers. Therefore, complementary treatments are needed to improve the impacts of chemotherapy agents. The effect of probiotics as cancer-preventing agents through involvement in the activation of apoptotic pathways has been established. The present study sought to investigate how the heat-killed form of Saccharomyces cerevisiae (as a probiotic) could affect the Akt/NF-kB-induced apoptosis in colon cancer cells, the SW480 cell line. The cytotoxic effects of heat-killed yeast (HKY) and 5-fluorouracil (5-FU, as a positive control drug) were assayed using the MTT method. Morphological changes followed by apoptosis were examined using DAPI staining. The transcription and translation level of apoptosis genes were explored with qRT-PCR and western blotting. The data were analyzed using GraphPad Prism V6.0 Software. The results showed that HKY could induce apoptosis in colon cancer cell line through downregulation of p-Akt1, Rel A, Bcl-XL, pro-caspase 3, and pro-caspase 9 expressions, and upregulation of BAX, cleaved caspase-3, and cleaved caspase-9. Besides, Akt protein expression was not affected. It is noticeable that HKY had a better modulating effect on BAX expression compared with 5-FU. It was able to modulate Akt/NF-kB signaling pathway followed by the apoptotic cascade.

Overview of Albumin and Its Purification Methods.

As the most frequent plasma protein, albumin constitutes more than 50% of the serum proteins in healthy individuals. It has a key role in oncotic pressure maintenance and it is known as a versatile protein carrier for transportation of various endogenous and exogenous ligands. Reduced amounts of albumin in the body will lead to different kinds of diseases such as hypovolemia and hypoproteinemia. It also has various indications in shocks, burns, cardiopulmonary bypass, acute liver failure and etc. Further applications in research consist of cell culture supplement, drug delivery carrier and protein/drug stabilizer. So, the demand for albumin increased annually worldwide. Due to different applications of albumin, many efforts have been accomplished to achieve albumin during a long period of time. In this review, an overview of serum albumin and different purification methods are summarized.

Purification and Characterization of Bovine Serum Albumin Using Chromatographic Method.

Purpose: Albumin is an abundant protein of blood and has many biopharmaceutical applications. The aim of this study was to purify bovine serum albumin (BSA) using produced rabbit anti-BSA antibody. Methods: The polyclonal antibody was produced against the BSA in rabbits. Then, the pure BSA was injected to three white New Zealand rabbits. ELISA test was done to evaluate antibody production. After antibody purification,the purified antibody was attached to CNBr-activated sepharose and finally it was used for purification of albumin from bovine serum. Western blotting analysis was used for functional assessment of immunoaffinity purified BSA. Results: The titer of anti-bovine albumin determined by ELISA was obtained 1: 256000. The SDS-PAGE showed up to 98% purity of isolated BSA and western blotting confirmed the BSA functionality. Purified bovine serum albumin by affinity chromatography showed a single band with molecular weight of 66 KDa. Conclusion: Affinity chromatography using produced rabbit anti-BSA antibody would be an economical and safe method for purification of BSA.