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Uptake studies in rat Peyer's patches,cytotoxicity and release studies of alginate coated chitosan nanoparticles for mucosal vaccination Olga Borges a,*, Anabela Cordeiro-da-Silva b, Stefan G. Romeijn c, Maryam Amidic,Adriano de Sousaa, Gerrit Borchard c,d, Hans E. Jungingerc,e Center for Pharmaceutical Studies, Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Rua do Norte, 3000-295 Coimbra, Portugal 指導老師：林鴻儒 教授 報 告 者：陳禹樵 民國九十八年十二月一日 1 Outline Introduction Material and methods Results and discussion Conclusions 2 Introduction 近年來投入藥物傳導系統的研究越來越 多，主要是因為它的生物相容性與具有 時間性的控制藥物釋放行為，使學術界 越來越多人投入研究。 因此本研究計畫開發新型系統，利用 Alginate包覆Chitosan，使卵白蛋白能順 利到達小腸淋巴系統釋放。 3 Material Chitosan Alginate Ovalbumin MTT trypan blue FBS polymers reagents 4 Methods 0.25wt% chitosan溶解在醋酸中 加入3.5 mL 10wt%硫酸鈉溶液至200 mL 離心30min 3500rpm 液態氮冷凍乾燥 0.25wt% ovalbumin 0.4wt% nanoparticles 浸泡在含有ovalbumin的PBS中120min 添加與nanoparticles等量的1wt% alginate 攪拌20min後，離心10min 1600rpm 浸泡在0.524 mM CaCl2的HEPES緩衝溶液10min 5 Fig. 1. In vitro release profiles of ovalbumin from uncoated chitosan nanoparticles into various aqueous dissolution media at 37 °C. Mean±standard deviation, n=3. 6 7 Fig. 3. Viability of splenocytes measured by the reduction of MTT. The cells were incubated for 20 h in the presence of two different concentrations of polymers, chitosan and sodiumalginate. The control group corresponds to the assay just with cells in culturemedium. Results represent themean±standard error of themean for 3 differentmice, each performed in triplicate (n=9). Statistical differences between control group and formulations are reported as: *p<0.05, **p<0.01. Cell viability (% of the control)=[A]test/[A]control×100. 8 Fig. 4. Viability of splenocytes measured by the reduction of MTT. The cells were incubated for 20 h in the presence of two different concentrations of the nanoparticles, alginate coated chitosan particles and uncoated chitosan particles. The control group corresponds to the assay just with cells in culture medium. Results represent themean±standard error of themean for 3 differentmice, each performin triplicate (n=9). Statistical difference between control group and formulations are reported as: *p<0.05, **p<0.01. Cell viability (% of the control)=[A] test/[A] 9 control×100. Fig. 5. The viability of splenocytes measured by counting trypan blue stained and unstained cells with hemocytometer. The cells were incubated for 15 h in the presence of two different concentrations of the polymers, chitosan and sodium alginate. The control group corresponds to the assay just with cells in culture medium. Results represent the mean±standard error of the mean for 3 different mice (n=3) and each count was the result of pooled samples from the same mice. No statistical differences were found between the control and the polymer groups. 10 11 Fig. 7. Flow-cytometric FL3 histogramoverlay of control groups on the left picture and control plus polymer treatment (sodium alginate and chitosan) on the right picture. 12 13 Conclusions 本研究證明chitosan奈米顆粒包覆具有醫療性蛋白是一 種溫和藥物載體，且具有很高的包覆率。 由實驗得知此型藥物輸送載體外層以alginate包覆可提 升穩定度，使藥物釋放可以達到控制。 由細胞毒性測試發現，此藥物載體沒有毒性，有利於 往後應用在人體方面。 體內研究與實際疫苗正在進行研究這一新的療效 粘膜給藥系統。 14 Thank you for your attention!! 15 卵白蛋白(ovalbumin) 卵白是一種具有黏性、異型的物質，約佔卵的 60%，其中卵白的蛋白質可分為卵白蛋白(54%)、 伴白蛋白(13%)、卵類黏蛋白(11%)、卵球蛋白 (3%)、溶菌酶(3.5%)及卵黏蛋白(2%) 其中卵白蛋白含量最高，且具有游離的硫化氫 基。 16