Résumé
Background: Targeted cancer therapies have played a great role in the treatment of malignant tumors, in the recent years. Among these therapies, targeted toxin therapies such as immunotoxins, has improved the patient's survival rate by minimizing the adverse effect on normal tissues, whereas delivering a high dose of tumoricidal agent for eradicating the cancer tissue. Immunological proteins such as antibodies are conjugated to plant toxins or bacterial toxins such as Diphtheria toxin [DT] and Pseudomonas exotoxin A [PE] . In this case optimizing and expressing Diphtheria toxin and Pseudomonas exotoxin A which their binding domains are eliminated play a crucial role in producing the desired immunotoxins
Materials and Methods: We expressed the truncated DT and PE toxin in a genetically modified E.coli strain BL21 [DE3]. For this reason we eliminated the binding domain sequences of these toxins and expressed these proteins in an expression vector pET28a with the kanamycin resistant gene for selection. The optimization of Diphtheria toxin and Pseudomonas exotoxin A expression was due to different IPTG concentration, induction and sonication time
Results: We observed that the optimal protein expression of the Diphtheria toxin was gained in 4 hours of 0.4 mM IPTG concentration at 25celsius on the other hand the optimization of Pseudomonas exotoxin A protein occurred in 4 hours of 0.5 mM IPTG concentration at 25 celsius
Conclusion: Our study also showed lower IPTG concentrations could result in higher protein expression. By optimizing this procedure, we facilitate the protein production which could lead to acceleration of the drug development
Résumé
The aim of this investigation was to design and develop nanoemulsions [NEs] as novel delivery systems for rapamycin. Phase behavior of quaternary systems composed of Traicetin [as oil], various surfactants and co-surfactants and water at different surfactant/co-surfactant weight ratios was investigated by the construction of phase diagrams. Formulations were taken from the o/w NE region of the phase diagrams, depending upon the extent of NE domain. The spontaneous emulsification method was used to prepare various formulations containing 1 mg/mL of the drug. The NEs were characterized and subjected to stability tests at various temperatures over 9-12 months. Cumulative drug release from the selected formulations was determined for a period of 48 h using a dialysis sac. The assay of rapamycin was carried out using an HPLC technique. The effect of NEs on the viability of SKBR-3 cells was evaluated by MTT assay. The integrity of Caco-2 cell monolayers was measured by Transepithelial Electrical Resistance [TEER] and the transport of rapamycin-loaded NEs across Caco-2 cell monolayers was then assessed. The uptake of NEs by SKBR-3 cells was also investigated using florescence microscopy. Maximum drug release was observed in case of 4 formulations prepared with Tween 80 and Tween 20. MTT test results revealed different toxicity of NEs for SKBR-3 cell line and TEER demonstrated that formulations containing Tween 20 caused a more considerable decrease in cell integrity in comparison with those prepared with Tween 80. The results obtained from cellular uptake experiments were in consistent with those obtained from TEER and cytotoxicity experiments