Preparation Of Protein-drug-loaded Thiolated-chitosan Microcapsules And Research Of It's Vitro Property

Objective:To explore the best preparation condition of different thiolated-degree protein-model-drug loaded chitosan microcapsules,research the influence of thiolated-degree on the appearance,particle diameter,encapsulation efficiency,drug loading and vitro release performance of these microcapsules,detect the safety of the thiolated-chitosan microcapsules used as drug deliver carrier,provide the usefull parameter for the animal experiment and clinical research of thiolated-chitosan microcapsules.Methods:(1)Used chitosan(molecular weight:50 kDa,deacetylation degree:96.2%) and thioglycolic acid as material,ethyl-dimethyl-amino-propyl carbodiimide(EDAC) as condensing agent to synthesis high-,middle- and low-thiolated degree chitosan derivates,used infrared spectroscopy method to do the qulitation detection,Ellman's method to do the quantity detection and elemental analysis for the production; (2)Used sodium tripolyphosphate(TPP) as cross-linking agent,bovine serum albumin as model drug to prepare the protein-drug-loaded thiolated-chitosan microcapsules by the method of ionotropic gelation,used encapsulation efficiency of the drug as evaluating indicator to explore the best preparation condition of the three thiolateddegree chitosan microcapsules by orthogonal design;(3) Prepared three kind of thiolated-chitosan microcapsules under the best preparation condition,observe the appearance of these microcapsules by scanning electrical microscope,detect the encapsulation efficiency and drug loading;(4) Determin the accumulated release rate of the microcapsules in the pH7.4,6.0,1.5 release medium within 12 hours;(5) Detect the cytotoxicity of the microcapsules by the method of MTT method.Result:(1) The infrared spectroscopy shows:to compare with the chitosan,the three synthesis products had new absorption peak near 1526 cm^-1 and within 2500 -2650 cm^-1,the former was the mark absorption peak of amide linkage,the latter was the mark absorption peak of the thiol group,they indicated that the mercapto-acetic acid was linked to the chitosan by amide linkage.(2) The quantity detection indicated that the amount of hydrosulfide were 46.18±2.8246μmol·g^-1,150.75±2.5558μmol·g^-1, 202.85±3.0520μmol·g^-1 when the addition of EDAC was 460 mg,1380 mg,2300 mg(ANOVA result:F=8207.746,P＜0.001,and P＜0.001 between the two groups, there were statistics difference ),the sulfur content were 0.17%,0.62%,0.71%.(3)The best preparation condition of the three kind of thiolated-chitosan microcapsules were: thiolated-chitosan /TPP(w/w) 4.2:1,pH value of the preparation system 5.4,the dropwise speed of TPP 20drops/min,the stir speed 200 rpm.(4) The microcapsules which were prepared under the best condition were globular shaped,the diameter were between 20-25μm,the drug loading were 32%-36%,the encapsulation efficiency were between 63.45%-45.97%depended on the difference of thiolated degree.(5) In the release medium of pH 1.5,the low-,middle- and high-thiolated microcapsules completed released after 7 hours,9 hours and 10 hours;In the release medium of pH 6.0,the low-thiolated microcapsules completely released after 9 hours,the accumulated release rate of middie- and high-thiolated microcapsules were 96.98±1.52%and 78.34±1.00%within 12 hours;In the release medium of pH 7.4,the low-thiolated microcapsules completely released after 10 hours,the accumulated release rate of middle- and high-thiolated microcapsules were 95.35±1.14%and 76.37±1.55%within 12 hours.(6) The relative proliferation of vitro HeLa cell after cultured with high-,middle and low concentration low-thiolated microcapsules for 24 hours were 81.86±1.04%,84.48±1.72%and 86.72±2.58%.84.63±2.53%, 88.85±2.81%and 91.88±4.50%for the middle-thiolated microcapsules.92.91±2.37%, 94.06±1.93%and 98.57±2.43%for the high-thiolated microcapsules.Conclusion:(1) Used EDAC as condensing agent,we can link the thioglycolic acid to the chitosan under the mild condition,the thiolated degree increase when the amount of EDAC increase.(2)The preparation method of thiolated- chitosan microcapsules are simple,the condition is mild,the factors in the preparation procedure have important influence on the encapsulation of the microcapsules.(3) When the thiolated degree increase,the dimeter of the microcapsules increase slightly,the encapsulation efficiency decrease,but the drug loading have no obvious difference.(4) The vitro release performance of the microcapsules are affected by the release medium and the thiolated degree of the chitosan,when the pH value of the release medium and the thiolated degree of chitosan increase,the release speed of the microcapsules decrease (5)To compare with the chitasan microcapsules which has been proved to be safe,the thiolated-chitosan microcapsules are safe and non-toxicity,they can be used as drug deliver carrier.