Optimization Of Preparing Chitosan Microspheres And Study On Its Acetylated Microspheres As Potential Chemoembolizating Materials

Transcatheter arterial embolization (TAE) is a medical technology that embolismmaterial is injected or sent to vascular by catheter in the X-ray and blocked bloodvessel so as to achieve the purpose of expected treatment. It could treat diseasesthrough clogging blood flow; reduce the blood supply of nidus or body parts. Owingto minimally invasive, image guide and selective target blood vessels catheterization,this technology make the accuracy and controllable of embolism greatly enhanced andbecome a revolutionary clinical treatment. The microspheres have many advantageson good effect in embolism, target ability in particular tissue and control release fromdrug encapsulation. Microsphere is the most common embolism carrier appliedwidely and attracts more and more attention.Chitosan, the N-deacetylated derivative of chitin, is a natural cationpolysaccharide. Chemical name is β-(1,4)-2-amino-2-deoxy-D-glucan. Chitosan isused in many field such as medicine, food, agriculture, biochemical, chemical,environmental protection and so on, due to its useful features such as biocompatibility,biodegradability, low toxicity, antibacterial activity, antitumor, enhance immunity andantioxidant activity, etc. In recent years, embolic agents are made from Chitosan andits derivatives have been focused by many researchers. It is not only an excellentbiological activity but also a drug carries, and have dual role in physical embolismand chemical treatment. The Chitosan microspheres (CMs) and acetylated Chitosanmicrospheres (ACMs) are prepared by emulsion cross-linking method. Thephysicochemical property and biocompatibility of microsphere are investigated by aseries experiment, and then ACMs are used for embolizing trial on animal, whichcould provide some useful references for further in vivo clinical applications.CMs are prepared by W/O emulsion cross-linking method using single factor analysis and response surface methodology (RSM). Meanwhile, the factors effectingCMs preparation are comprehensive survey such as Chitosan concentration, aceticacid concentration, Span80volume, toluene volume, stirring speed, emulsified time,formaldehyde volume and cross-linking time. These factors levels are optimized byPlackett-Burman design, the steepest ascent experiment and Box-Behnken design.The best preparation conditions for CMs via many experiments are shown as follows:2%(w/v) Chitosan dissolved in1.7%(v/v) acetic acid, and then100ml Chitosansolution were added into488ml toluene with7ml Span80and2ml tween-60, themixture were stirred at1100rpm for60min,10ml formaldehyde solution was addedto the system with continuous stirring for60min. The microspheres with smoothsurface are prepared, thus, the maximum predicted value of CMs (120mesh-60mesh)(7.93g) and experimental value (8.03g) are not significant.ACMs with smooth surface and good dispersion are made by acetylating CMs.The characteristic absorption peak of-NHCOCH3groups appear in1595cm-1asshown in FT-IR spectra. The degree of deacetylation (DD) of Chitosan, CMs, ACMsis90.57%,84.83%and20.92%, respectively. It is showed that5.7%of amino iscross-linked in CMs, and then63.9%is acetylated in ACMs. Swelling rate (SR) ofCMs varies inversely as the pH value but less influence for ACMs. While, SR of CMsand ACMs is more effected by environmental temperature, increasing with risingtemperature, otherwise decreasing. The structure of CMs/ACMs remain stable afterautoclaving at121℃,150kPa for1h, the morphology of microsphere is perfectafter3month under room temperature. The degradation rate of ACMs is faster thanCMs in lysozyme, the mass decreased by58.1%and40.7%, respectively. Some cavityand uneven surface appears on microsphere after degradation.The amount of adsorbed protein on CMs is more than on ACMs and also longerin the time of adsorption equilibrium, due to more amino group in CMs. Hemolysisrate (HR) of ACMs in10mg/ml and50mg/ml all less than5%, but obvioushemolysis for CMs in50mg/ml (HR>5%). Blood clots formed reaction on ACMs islight, and the weight of blood clots on ACMs is less than on CMs, it is showed thatACMs has good blood compatibility avoid hemolysis than CMs. Cytotoxicity of CMs/ACMs to mouse embryo fibroblasts (MEFs) proliferation is investigated byMTT method, the results shows that MEFs successfully proliferate on microsphere(132μm and260μm) but not on microsphere (429μm). Apparently, relative growthrate (RGR) of MEFs for CMs is decrease with incubated time but increase with timefor ACMs. Therefore, it is showed that ACMs have better haemocompatibility and cells compatibility via protein adsorption, blood compatibility and cytotoxicityexperiments.Biological safety in vivo of CMs/ACMs is evaluated. Extract of material have nohypersensitized and hormesis effect on skin and cornea of rabbit. Hormesis of tailintravenous injection (i.v) with high doses (0.5ml/10g) is slightly stronger thanintraperitoneal injection (i.p) to mice. The weight drops after24h after i.v and thenrecovers increase, and it is keeping increase after i.p, the results shows that extract ofmaterial have no potential toxicity. The liver and kidney function of rat is normal after3d,7d and14d as CMs/ACMs is implanted in muscle, but levels of white blood cell(WBC) rise after3d and7d (p<0.05) due to inflammatory response by implanting andthen return to normal. Microspheres (CMs/ACMs) are gradually degradated in vivo,and has good histocompatibility because of no causing significant tissue rejection.Rabbit ear artery is embolized with ACMs for3d, inflammation and edema in ear,scabby phenomenon in ear tip because of vascular barrier. After7d, inflammation andedema disappear in ear, there is obviously scabby, ischemic necrosis in ear tip. After15d, arteriole atrophy disappears in ear tip, surrounding tissues is coagulation necrosisand partly fall off, edge skin border with necrotic tissue become atrophy andthickening. The result shows that ACMs have an obvious effect on vesselembolization.Loading efficiency (LE), loading content (LC) and control release behaviors invitro of doxorubicin hydrochloride (ADM HCl) loading microspheres(ADM HCl-CMs/ACMs) are evaluated in this study. The LE and LC of CMs/ACMsare also about54.8±1.23%,11.1±0.61%, moreover, the LE and LC of undried CMshigher than dried CMs (62.53%,13.67%, respectively) because of dehydration. Thedynamic dialysis method is employed to study in vitro control release behaviors of ADM HCl-CMs/ACMs. Release rate of ADM HCl in pH4.0medium is faster than inpH7.0medium. Comparing to CMs, ACMs shows an obviously controlled releasestate in the experiment and has some advantages in the drug releasing.It is found that CMs, especially ACMs have advantages in haemocompatibility,cytocompatibility, histocompatibility, high biological safety, and effectiveembolization, could be used as a potential vascular embolic material for clinicalapplication.