In vitro co-delivery evaluation of novel pegylated nano-liposomal herbal drugs of silibinin and glycyrrhizic acid [nano-phytosome] to hepatocellular carcinoma cells

Objective: This study aimed to evaluate a co-encapsulated pegylated nano-liposome system based on two herbal anti-tumor drugs, silibinin and glycyrrhizic acid, for delivery to a hepatocellular carcinoma [HCC] cell line [HepG2]

Materials and Methods: In this experimental study, co-encapsulated nano-liposomes by the thin layer film hydration method with HEPES buffer and sonication at 60% amplitude. Liposomes that co-encapsulated silibinin and glycyrrhizic acid were prepared with a specified molar ratio of dipalmitoylphosphatidylcholine [DPPC], cholesterol [CHOL], and methoxy-polyethylene glycol 2000 [PEG2000]-derived distearoyl phosphatidylethanolamine [mPEG2000-DSPE]. We used the MTT technique to assess cytotoxicity for various concentrations of co-encapsulated nano-liposomes, free silibinin [25% w/v] and glycyrrhizic acid [75% w/v] on HepG2 and fibroblast cell lines over a 48-hour period

Results: Formulation of pegylated nano-liposomes showed a narrow size distribution with an average diameter of 46.3 nm. The encapsulation efficiency [EE] for silibinin was 24.37%, whereas for glycyrrhizic acid it was 68.78%. Results of in vitro cytotoxicity showed significantly greater co-encapsulated nano-liposomes on the HepG2 cell line compared to the fibroblast cell line. The half maximal inhibitory concentration [IC50] for co-encapsulated pegylated nanoliposomal herbal drugs was 48.68 microg/ml and free silibinin with glycyrrhizic acid was 485.45 microg/ml on the HepG2 cell line

Conclusion: This in vitro study showed that nano-liposome encapsulation of silibinin with glycyrrhizic acid increased the biological activity of free drugs, increased the stability of silibinin, and synergized the therapeutic effect of silibinin with glycyrrhizic acid. The IC50 of the co-encapsulated nano-liposomes was lower than the combination of free silibinin and glycyrrhizic acid on the HepG2 cell line

Anticancer effect of the IgY that produced against a small peptide with 15 amino acids of human DR5 on MCF7 cell line

Tumor necrosis factor- alpha [TNF-alpha] plays an important role in diverse cellular events such as septic shock, induction of other cytokines, cell proliferation and apoptosis. Tumor necrosis factor [TNF]-related apoptosis-inducing ligand [TRAIL] is currently attracting great interest as a potential anticancer drug. TRAIL could selectively induce apoptosis in tumor cells in vitro and in vivo by a death receptor-mediated process. TRAIL shows a high degree of promiscuity as it binds to the DR5 receptor and it is generating considerable interests as a possible anticancer therapeutic agent. Use of TRAIL or its antagonist could be a good anticancer treatment in future. The extracellular domain of DR5 human protein which has the attachment part of this ligand to TRAIL ligand is considerable domain of it. We produced a small peptide with just 15 aminoacids from this domain, with peptide synthesizer. Then inject them to hens to immunize them and achieve high affinity IgY. At least, obtained IgYs specially recognize DR5 protein and in vitro start exclusively to induce death in the MCF7 cell line, and interestingly not on normal cells

Evaluation of cisplatin and cisplatin-loaded magnetic iron oxide nanoparticles on BCL2 and BAX genes in the breast cancer T47D cell line

Breast cancer is the second leading cause of cancer death in women. Cisplatin is a traditional cancer drug commonly used in chemotherapy for killing cancer cells. Modulation at the mRNA levels of apoptotic related genes often correlate with the sensitivity of various types of cancer cells to chemotherapeutic agents. Nanoparticulate drug delivery systems are being developed to effectively deliver smaller doses of chemotherapeutic agents and control drug distribution in the body. In this study, we evaluate the expressions of BCL2 and BAX genes in T47D treated with cisplatin and cisplatin nanoparticles, which can result in a new approach to breast cancer therapy. In this study, we treated T47D cells with different concentrations of cisplatin and cisplatin nanoparticles at 48 h. The IC50 was determined. We extracted RNA by using RNX solution, after which cDNA was synthesized. The precise primers for the BCL2, BAX and TBP genes were designed by specific software. The quantity of BCL2 and BAX gene expression compared to TBP gene [reference gene] was analyzed using real-time PCR. BCL2 and BAX gene expression levels in T47D cells treated by cisplatin were 0.7 [BCL2] and 1.48 [BAX], in T47D cells treated with cisplatin-loaded nanoparticles, the gene expressions were 0.03 [BCL2] and 2.41 [BAX]. In this study, the results have shown that cisplatin-loaded nanoparticles are effective anticancer agents. Cisplatin nanoparticles induce apoptosis in human breast cancer cell lines. We have shown that cisplatin nanoparticles strongly increased cytotoxicity in comparison to the free drug in the T47D cell line

Cloning of the corynebacterium glutamicum ddh gene in order to increasing the lysine production

Due to wide range of application of L-Lysine, as an essential amino acid, in different industries, the demand for this amino acid has been increased and this has been led to the development of research on industrial production of L-Lysine. In this study, we tried to increase the yield of production of L-Lysine by genetic optimization. After inspection of different effective enzymes in Lysine biosynthesis pathway, Diaminopimelate dehydrogenase [EC 1.4.1.16] enzyme was selected. This enzyme would be coded by ddh gene. After chromosomal DNA extraction of C.glutamicum ATCC 21799 and designing of primers, the gene fragment was separated from genome by PCR withpfu DNA polymerase enzyme and was cloned in TAcloning vector for facilitation of cloning and determination of nucleotide sequence. Then, enzymatic digestion of TAcloning vector and pET28a vector were performed by EcoRI and Sail restriction enzymes which their products were ddh gene and linear vector. Ligation reaction was accomplished and for checking the accuracy of ligation, it was transformed into E.coliDH5a and finally in expression host, E.coli BL21 [DE3]. The 1150bp band in PCR products and enzymatic digestion of extracted vectors with EcoRI and Sail, sequencing and SDS-PAGE confirmed the accuracy of cloning. Recombinant bacterial colonies were investigated and confirmed by two methods [PCR and enzymatic digestion]. This study showed significant increased expression rate of DAP dehydrogenase enzyme in this expression vector for the first time

[Up regulation of CD82 gene in prostate cancer PC-3 cell line treated with silibinin]

Prostate cancer is one of the most common cancer in the developed countries. Most of cancer deaths are due to development of metastasis. Hence, prevention of metastasis is critical. Silibinin is a flavonoid component that inhibits cell proliferation and causes cell death of human prostate cancer. In this study, the expression of CD82 gene in PC-3 cells treated with escalating concentrations of silibinin was evaluated which can result in new view for prostate cancer therapy. In this study, PC-3 cells were treated with different concentrations of silibinin for 24h. The LD50 was determined. RNA was extracted by trizol, then cDNA was synthesized. Precise primers were designed for CD82 and GAPDH genes by specific software. Quantity of CD82 gene expression compare to GAPDH gene in different concentrations of silibilin was analyzed using very sensitive quantitative Real-time PCR. CD82 gene expression in PC-3 cells treated with 100, 150 and 200?g/ml of silibinin at 24h was increased by 1.97 +/- 0.26 [P<0.05], 3.00 +/- 0.26 and 3.43 +/- 0.43 [P<0.01], respectively. The results of quantitative Real-time PCR indicated that silibinin can probably decrease metastasis, by up-regulation of CD82 metastasis suppressor gene in PC-3 cells

Isolation and purification of rat islet cells

Background and Purpose: It is thought that transplantation of islet cells would cure diabetic patients in world. Islet cells transplantation in people suffering from diabetes is of technical interest. A standardized procedure was developed for the preparation of rat islet cell grafts for purification of islet cells

Materials and methods: In this process, after collagens digestion of pancreases, islets were isolated and dissociated, then with enzymatic procedure by DNase and trypsin, the islet cells changed in to single cells and these cells were assayed by flow cytometery

Results: Flow cytometery of these cells indicated that there were 91% of beta cells in cell suspension. Most of the exocrine particles were lost during digestion

Conclusion: Purified endocrine islet cell grafts were prepared by pure beta-cells, with or without endocrine non-beta cells. The purified aggregates were devoid of non-endocrine cells and damaged cells

Preparation and characterization of theophylline-chitosan beads as an aapproach to colon delivery

Chitosan with excellent biodegradable and biocompatible characteristics has received attention as an oral drug delivery vehicle for controlled-release formulations. In this study an enteric-coated capsule containing theophylline-chitosan beads based on 2[3] factorial designs was prepared as a colon drug delivery system. The theophylline-chitosan gel beads were formulated by adding the drug-containing solution of chitosan into tripolyphosphate solutions, dropwise. The obtained beads were washed with water and freeze-dried before filling into the capsules. Eudragit[R] S100 was then used to enteric-coat the prepared capsules. Drug entrapment efficiency and the effects of different variables including: bead morphology, swelling behavior of the beads and the release behavior of the system on these parameters were investigated. Results showed that the highest and lowest swelling ratio is obtained at pH 4.5 and 7.2, respectively. These studies have shown that chitosan concentration and drug polymer weight ratio significantly affect the drug entrapment. Decreasing the drug solubility in external phase caused a significant increase in drug loading. External phase saturation with theophylline and tripolyphosphate, as well as decreasing temperature, have increased drug loading. Furthermore, the lowering of temperature had a significant effect on bead's hardness. The release of theophylline from freeze-dried beads filled in enteric-coated capsules was also investigated. Release of theophylline was prolonged with saturation of both drug and tripolyphosphate in the external phase. Results showed that the release of theophylline from chitosan beads is possibly due to more than one mechanism, possibly dissolution, diffusion and relaxation of the polymer chains

Effects of protoplast fusion on S-endutoxin production in bacillus thuringiensis Spp. [H14]

In this study, mutant forms of Bacillus thuringiensis spp. israelensis [H14] were produced. These mutants were identified when the cells were cultured on chloramphenicol plates and stained with crystal violet. Protoplasts of the mutants were isolated by enzymatic digestion [lysozyme] of the cell walls at the presence of an osmotic stabilizer. The protoplasts were induced to fuse to each other in the presence of PEG 6000. The frequency of regeneration and recombination was 80% and 2 10-4, respectively. In order to survey the effect of protoplast fusion on production of toxin, anti-serum against pure toxin was raised in rabbit and was used in single radial immunodiffusion. The comparison of -endotoxin concentration between B. thuringiensis fusion and the wild type strains showed that B. thuringiensis fusion has 1.48 time more toxin than wild type