Preparation Of Alginate Microspheres And In Vitro Releasing Behavior Of Encapsulated Bovine Serum Albumin

More and more therapeutical protein drugs are being developed by means ofrecombinant DNA technique, but the clinic applications through oral delivery systemsare still not successful for most of them, due to their short half-life resulting fromacid-catalytic or proteolytic degradation in the gastrointestinal tracts. A potentialsolution is microencapsulation through which the protein drugs are encapsulated,preventing from the degradation, and releasing in controlled manners. Alginatemicrosphere seems very attractive to be used as such a drug delivery carder becauseof its convenience in gel formation and good biocompatibility. And the alginatemicrospheres with different ranges of diameters can be prepared by a variety ofcommonly used techniques. However, many technical challenges still exist about howto develop delivery systems to improve their properties and control their releases invivo, such as controlling the diameter of the microspheres, improving the loadingcapability and preserving the therapeutical effectiveness of protein drugs.In this dissertation, bovine serum albumin (BSA) was chosen as a protein drugmodel and a natural biodegradable polysaccharide (alginate) was used as theencapsulation material. The influences of processing parameters of different methodsincluding dropping, spraying and emulsification on the performances of alginatemicrospheres (such as diameter, encapsulation efficiency, loading capability andrelease profiles) and the activities of BSA contained in microspheres wereinvestigated systematicly. At the same time, the preparation processes was improvedtechnologically. And the feasibility of the application of microspheres containingprotein drugs prepared by different methods on oral administration of proteins wasfurther studied in detail.The encapsulation of BSA with the alginate microspheres prepared by a sprayingtechnique was firstly evaluated. Under the optimized conditions, the mean diameterof the alginate microspheres was 70μm, with a good spherality. The encapsulationefficiency of BSA increased with the decrease of the pH value in CaCl₂ solution, anda BSA loading capacity of 8% (w/w) was obtained, without noticeable effect on themolecular structure and antigenicity of the encapsulated BSA. The in vitro release of BSA showed a controlled release behavior owing to the excellent pH responsiveproperties of alginate microspheres. The BSA release rate was slower in simulatedgastric fluid (SGF), but significantly increased in simulated intestinal fluid (SIF). Thegas flow rate applied to the manufacture of the alginate microspheres also affected therelease behavior of BSA, with a slightly parallel increase. A three-step releasebehavior of BSA was observed in a simulated usual gastro-intestinal transit. Onlyabout 5% of BSA was released into SGF within 2h, indicating that alginatemicrospheres are useful in protecting protein drugs from degradation in a gastricenvironment.When electrostatic field was applied to the spraying process (SEF), the diameterdistribution of the alginate microspheres was narrowed, with a significantimprovement in the spherality. Comparing with the alginate microspheres obtained bythe spraying without electrostatic field effect, the diameter of the microspheresprepared decreased about 20%, as well as the encapsulation efficiency and loadingcapacities increased about 6% and 30%, respectively. The molecular structure of theencapsulated BSA was not affected by the preparation and release process. Moreimportantly, the BSA release behavior from the microspheres prepared by SEF wasmore controllable, and the total release amount of BSA increased with the increase ofthe voltage.Soybean salad oil, which was widely used in food industry, was chosen as an oilphase for preparing alginate microspheres by the emulsification technique, with anultrasonic pretreatment. The alginate microspheres with an average diameter of 10μmwere obtained under appropriate conditions. The encapsulation efficiency and loadingcapability were 70% and 4% (w/w), respectively. As the concentration of ALGincreased and the amount of BSA decreased, the encapsulation efficiency increased,but the loading capability decreased, without significant effect on the molecularstructure of the encapsulated BSA owing to an improvement in the homogeneousemulsion by ultrasonic sound, as well as the protection of high viscous soybean saladoil to the BSA.According to the drug release mechanism for swellable alginate matrix, a first-orderkinetic model was postulated and modified to describe the release behavior of BSAfrom the alginate microspheres prepared by the aforementioned three methods. Theequation fitted the release data well when an obviously initial burst release existed andcomposition of release media changed. In a uniform medium, the rate constant was inan inverse ratio to the diameter of alginate microspheres. Increasing the gas flow rate of the spraying, the stirring speed of the emulsification process, and the electricvoltage of the electrostatic field decreased the mean diameter of the microspheressignificantly and enhance the release rate of BSA. On the other hand, the release rateconstant was larger in SIF than in SGF owing to their different drug releasemechanisms.In order to improve the mechanical strength of the microspheres and extend therelease period of the encapsulated BSA, polyvinyl alcohol (PVA), polyethylene glycol(PEG), starch, and chitison (CHI) were selected as surface complex materials, and theperformance of the modified alginate microspheres were investigated. It was foundthat the encapsulation efficiency increased for all these microspheres, theencapsulation efficiency of three-components (ALG-PVA-CHI) microspheres wasmore than 80%, the breakage ratio was less than 3% over 24 hours, and the releasetime of the BSA was doubled. It was also showed that these surface complex materialshad no effect on the molecular structure of the encapsulated BSA.Given the significant impact of the diameter on the performance of themicrospheres, the influences of the main operating parameters in dropping, droppingwith electrostatic field (DEF), spraying and SEF on the size distributions of thealginate droplets were studied. The formation mechanism of the alginate dropletsprepared by dropping or DEF could be categorized into two different modes: drippingand jetting modes, which were controlled by the liquid flow velocity and the voltageapplied to the system. By employing an effective force analysis, the data in each modeshowed to be well agreed with numerical simulations within 7% deviation. And theliquid flow velocity played a more important role on the mean diameter of the alginatedroplets in dripping mode than in jetting mode. Then, a Gretzinger equation was usedto simulate the diameter of the droplets obtained by the spraying method. Thedecrease in the diameter was more sensitive to the increase of the gas flow rate than tothe decrease of the liquid flow rate, and the predicted results of the two models fittedwell with the experimental data.Finally, the BSA recovery from the waste alginate microspheres was exploited,expecting to improve the economic performance of the processes. A solution wasdeveloped to break up the microspheres not suitable for drug delivery use and dissolvethe BSA contained. The solution was then salted-out by ammonium sulfate (AS)precipitation, and the obtained deposition was purified by gel filtration. It was foundthat under 75% AS saturation at 4℃for overnight or 70% AS saturation with 3 minultrasonic treatment for 1h, the recovery yields of BSA from CaCl₂ solution and microspheres-broken solution were as high as 80% and 50%, respectively. ThroughSDS-PAGE and gel filtration, the purified sample was obtained and the purity of therecovered BSA reached 98%, indicating that a higher utilization efficiency of proteindrugs and a lower cost of the drug delivery system can be developed.