| The supply of oxygen is a limiting factor in the production of fermentation products. Adequate oxygen supply is the key of stabilizing the fermentation, increasing the output and decreasing the cost. Traditional oxygen supply methods, such as increasing ventilation, demand high energy-consuming and expensive equipment. In the late1970s, the finding of Vitreoscilla hemoglobin (VHb) promoted the development of industrial fermentation of antibiotics and other drugs. The transfer of Vitreoscilla hemoglobin gene (vgb) into the host cell by means of genetic engineering can greatly improve the utilization of oxygen, and thereby enhancing the yield and saving the cost.Nanotechnology is the technology using individual atom or molecule to prepare materials with the structural dimensions in the range of0.1to100nanometers and study the quality and application of the materials. Nanomedicine is a new class of drug formulations prepared by nanotechnology and nano-carrier, among of which, nano-micelle has drawn great concern in the delivery of insoluble drugs. Micelles have the characteristics of high drug loading, high stability, simple preparation process, diversity carrier, degradation in vivo, strong transmembrane ability and antagonizing epithelial intestinal p-glycoprotein, etc. It is considered to be the main research direction of improving the oral bioavailability of poorly soluble drugs in the future.In this study, we have constructed one kind of genetically engineered tacrolimus producing bacteria with vgb, synthesized a novel nano-carrier material, prepared nano-micelles and studied it’s pharmacological properties. All of these results layed a foundation for the development of materials and new formulations, as well as the future applications of tacrolimus. The main research methods and results are as follows.1Construction of genetically engineered bacteria with vgb and the influence of vgb on the yield of tacrolimusFirstly, the production of tacrolimus by Streptomyces tsukubaensis was improved by conventional UV mutagenesis techniques. The UV mutagenesis conditions are: spore concentration of107～108CFU/mL, UV irradiation using a15W UV lamp for100s in a distance of20cm, and the lethality over90%. Canidia albicans was used as the indicator in double-layer plate bacterial inhibition test and HPLC was used to determine the yield of tacrolimus. We obtained two positive mutation strains, one of which could increase the yield of tacrolimus by27.7%. Then, the vgb gene was integrated into the genome of the tacrolimus-producing strains to further improve the yield of tacrolimus. The vgb gene, encoding VHb, was integrated into the chromosome of Streptomyces tsukubaensis to alleviate oxygen limitation and enhance the yield of tacrolimus. The VHb-specific spectrum was observed for the recombinant strain by CO-difference spectrum analysis. Compared to the original, the yield of tacrolimus produced by the strain bearing vgb increased2.0-fold under oxygen-limited condition. Last, the fermentation medium and conditions were optimized. The optimal fermentation medium consisted of glucose25g/L, dextrin80g/L, soybean meal10g/L, soya peptone10g/L, and glycerol10g/L. Based on the optimal fermentation medium, the seed age and inoculation amount were optimized as: seed age of20h and inoculation amount of2%. Under this conditions, the production of tacrolimus could reach to127.78-130.31mg/L. These results provide an effective strategy for improving the yield of tacrolimus and other antibiotics production.2Preparation, characterization and in vitro release study of MPEG-PLA/FK506micellesThe MPEG-PLA copolymers with different block ratios were synthesized via a ring opening polymerization of D, L-lactide (LA) initiated by MPEG using stannous octoate as the catalyst. After screening, the copolymer MPEG5000-PLA5000with the block ratio of1:1was found to be the best one which could form better micelles than other coploymers after loading tacrolimus. Tacrolimus was encapsulated into MPEG-PLA block copolymer using a double emulsion-solvent evaporation technique. The formula and process were optimized with homogeneous design-response surface methodology. The best formula is4.5%MPEG-PLA tetrahydrofuran solution as the organic phase,5mg of tacrolimus, and oil/water ratio of1:20. The micelles were spherical appearance, in line with core-shell structure. The micelles were monodisperse (PDI=0.100±0.023) with a mean particle size of90.5±.5nm, drug loading of9.5%and encapsulation efficiency of98.8%. Compared with FK506capsule, in vitro release profile showed that FK506/MPEG-PLA nanoparticles exhibited sustained release in the Higuchi equation, mathematical model, with the correlation coefficient up to0.9992. In vitro experiments showed that HEK293cell viability was over70%with MPEG5000-PLA5000’s concentration of1000μg/mL, indicating that the carrier material was safe and no toxic to the body.3Pharmacokinetics study in mouse and pharmacodynamic study in liver transplantation rat of MPEG-PLA/FK506micellesUsing FK506capsules as the control, we studied the pharmacokinetics of MPEG-PLA/FK506micelles in rats, established rat liver transplantation model, and carried out the pharmacodynamic study. Compared with oral FK506capsules, MPEG-PLA/FK506micelles showed faster absorption and better bioavailability. The Tmax shifted from3h to1h. The half-life was prolonged from13.698h to16.573h, the mean residence time were prolonged from23.593h to24.044h, and the AUC increased from508.251ng/mL-h to886.703ng/mL·h. All of these results proved the action of enhanced absorption, improved bioavailability and sustained release of micelles. MPEG-PLA/FK506micelles could prolong the survival time of liver transplantation rats. Compared with control, rats administered with MPEG-PLA/FK506micelles had a lower aminotransferase and bilirubin level. The liver pathology results showed that MPEG-PLA/FK506micelles were better able to fight against liver damage caused by rejection reaction and play a better liver protective effect. In summary, the constructed genetically engineered Streptomyces tsukubaensis with vgb can significantly improve low dissolved oxygen in the fermentation process, and provides a new way for improving the yield of FK506. The MPEG-PLA/FK506have the characteristics of simple preparation process, uniform particle size distribution, high drug loading, stableness, rapid absorption, sustained release, high bioavailability and good biocompatibility. So, it provides a new strategy and new method for enhancing the oral bioavailability of poorly soluble drugs. |
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