| In recent years, extensive attention has been paied to a very promising drugcandidate, curcumin, in anti-oxidant, anti-inflammatory, antitumor areas. Nonetheless,its drug development is limited for its poor stability, poor water-solubility and lowbioavailability. Therefore, many technologies such as micro-emulsion, cyclodextrininclusion, have been put forward. But using gel microspheres drug delivery systemhas not been reported. So a new DDS, Semi-IPN gel microsphere composed ofHPMC and Gelatin, has been raised to solve the problem.Firstly, curcumin analytical method in vitro has been established by UV withmax absorption wavelength of 421nm and linear absorption from 0-10ug. Thisanalytical method was confirmed to meet C.P. For curcumin, higher light intensity,temperature and strong acidic or alkaline condition leds to poor stabilty. Solubilitytests results show that the solubility in water and cyclohexane are low, but increasegreatlyin HPMC solution, reaching to 1027μg/mL.Secondly, the reverse suspension polymerization process of preparation wasdetermined, namely GSM is selected as dispersant (0.6g/30mL cyclohexane), stirringspeed equals to 500rpm and O/Wratio is 3:1. Nine formulations were successfulpreparaed. Semi-IPN structure formation was confirmed using FT-IR. The appearanceresults show that the microspheres are spherical with smooth surface, and averageparticle size is 201um with narrow distribution. Additionally results show that withthe increase of crosslinking agent and decrease of the content of HPMC, balancebibulous magnification and bibulous rate decline while mechanical strength increases.Thirdly, curcumin loaded microsphere was prepared. By using scanning electronmicroscope and optical microscope, the microspheres are spherical with smoothsurface, and average particle size is 216um.Characterized by FT-IR, the drug issuccessfully incroprated into microspheres. Stability test results show that, thestability of curcumin in microsphere is improved compared with the raw curcumin.Amorphous form mainly of curcumin in microsphere was observed through the X-rayRD and DSC, but the crystals also exist.Fourth,the effect of HPMC/gelatin ratio and crosslinking agent dosage oncurcumin release in vitro was also investigated. Results show that with the dosageincrease of HPMC and decrease of crosslinking reagent, encapsulating ratio decreases and solubility increases. The effective diffusion coefficient D was fitted usingHiguchi's mode and with the increase of HPMC and decrease of crosslinking agent,,effective diffusion coefficients rise.Finally, analytical method in vivo has been established by HPLC. Results showthat methodological verify complies with the CP. The drug loaded microsphere wasoral administration and blood was taken by angulus oculi medialis. PK parameterswere calculated. Results show that the absorption behavior is one compartment model.Bioavailability relative to raw curcumin is improved by4.863±1.185 times.To sum up, by HPMC-Gelatin semi-interpenetrating gel microspheres,curcumin's solubility, stability and bioavailability are improved and this newformulation will probably contribute to other poorly-soluble drugs to solve theseproblems.(Part II)In order to solve the problem of transferring NGF through blood brain barrier,the fusion protein composed of NGF and OX26 antibody was constructed by meansof genetic engineering. NGF-? gene was amplified and subcloned into pXA6 by PCRmethod, then the plasmid was transfected into COS-1 cells, and the fusion protein wastransient expressed following collecting the supernatant. OX26-NGF-? waspurified byProtein A affinity chromatography. Concentration was determined by the method ofFc ELISA, purity was dtermined by SDS-PAGE, at last it was confirmed that thefusion protein was constructed successfully combined with Western Blot analysis.
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