| Abstract: | 本研究以帶正電之幾丁聚醣 (chitosan，CS，去乙醯度≧85%) 及帶負電之三聚磷酸鈉 (sodium tripolyphosphate，TPP)，包覆鈣（calcium)及維生素D3 (vitamin D3) 製成奈米顆粒，以包覆與保護鈣及維生素D3之生理活性為目的。首先以臭氧（O3）將CS (271 kDa, Original Mw)氧化降解至200-250 kDa (High Mw)、100-150 kDa (Medium Mw)以及50 kDa以下(Low Mw)之分子量，分別溶於至濃度為2 mg/mL並將調pH值至4.7，接著，將不同濃度 (100、200、300、400、500、600、700、800、900及1000 mg/mL) 之Ca溶液及不同濃度（0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5及5.0 μg/mL))之Vit.D3溶液與上述CS溶液各別均勻混合後，滴入TPP (pH 9.0) 溶液使CS/TPP重量比為3/1、4/1、5/1及6/1攪拌(150 rpm,15 min)，製備幾丁聚醣-三聚磷酸鈉-鈣-維生素D3 (CS-TPP-Ca-Vit.D3) 奈米顆粒，並探討：Ca-Vit.D3濃度、CS分子量、及CS/TPP重量比對CS-TPP-Ca-Vit.D3奈米顆粒之包覆率、粒徑大小、表面電位及其奈米顆粒於模擬胃、腸液中之安定性。另外，以掃描式電子顯微鏡(SEM)照相觀察其奈米顆粒。結果顯示，在Ca及Vit.D3包覆率而言；Ca及Vit.D3濃度影響方面，Ca濃度約700 mg/mL時，Ca之包覆率較低，大或小於此濃度，其包覆率均增加，在55.6~74.3%之間﹔而Vit.D3之包覆率其濃度在0.5~5μg/mL間均未影響，維持在100%。CS分子量之影響方面，Original Mw、High Mw、Medium Mw及Low Mw者對鈣包覆率分別為70.27、68.37、64.01和64.48%，而Vit.D3之包覆率未受CS分子量之影響，均為100%﹔CS/TPP重量比之影響方面，其3/1、4/1、5/1及6/1者對鈣包覆率分別為64.48、70.64、74.12及72.77%，Vit. D3之包覆率均為100 %。CS-TPP-Ca-Vit.D3奈米顆粒粒徑而言， Vit. D3及Ca濃度比自0.5/100增至4/800μg/mg時，其粒徑自360 nm增至580 nm，但其濃度比再增至5/1000μg/mg時，其粒徑反而減小至495 nm；而隨著CS分子量降低，粒徑也跟著變低(1100 nm-500 nm)﹔另外，CS/TPP重量比增加時，數值上其差異並不大（502 nm增加至546.6 nm)。表面電位而言，Vit. D3及Ca濃度比之影響不大，其變動在+18.5~+22.5 mV間﹔隨著CS分子量降低， 其表面電位稍有減少之趨勢（+28.1mV~+24.1mV)。另外， CS/TPP重量比增加時，其奈米顆粒之表面電位呈現增加之趨勢（+24.1mV~+34.6 mV)。CS-TPP-Ca-Vit.D3奈米顆粒在模擬胃及腸液之安定性方面，若以Low Mw CS、CS/TPP：6/1及Vit.D3 /Ca/濃度：5/1000 μg/mg製備奈米顆粒後，置於pH 1.2及含pepsin (模擬胃液)，震盪2小時，調至pH 7.4並注入pancreatin (模擬腸液)繼續震盪6小時，發現，於8小時Ca及Vit.D3之釋放率分別約0.8%及16%，顯然很安定。
Chitosan(CS, original Mw 271 kDa, deacetylation ≧85%) and its hydrolateds (high Mw 200~250 kDa, medium Mw 100~150 kDa and low Mw≦ 50 kDa) were dissolved at a 2 mg/mL concentration and adjusted to pH 4.7,then individually mixed with calcium (Ca) solution and vitamin D3 (Vit.D3) solution at different concentration of 100~1000 mg/mL and 0.5~5.0 mg/mL, respectively. The mixed solution were added dropwise with sodium triployphosphate (TPP) solution (pH 9.0) at different CS/TPP mass ratio of 3:1, 4:1, 5:1 and 6:1 under stirring(150 rpm/15 min) to prepare CS-TPP-Ca-Vit.D3 nanoparticles. Such effective factors as Ca/Vit.D3 concentration ratio, CS molecular weight and CS/TPP mass ratio on the Ca and Vit.D3 encapsulation efficiency, particle size, zeta potential of the nanoparticles were investigated. Stability of the nanoparticles in the simulated gastric (pH 1.2,with pepsin) for 2 hrs and intestine (pH 7.4,with pancreatin continued for 6 hrs) system were determined. SEM of nanoparticles was also observed. As a result, firstly, on the Ca and Vit.D3 encapsulation efficiencies. The Ca encapsulation showed the lowest level at a Ca concentration of 700mg/mL, became increasing in both of using smaller or larger Ca concentrations and gave a range of 55.6~74.3%. The Vit.D3 encapsulation was 100% in all of Vit.D3 concentration. CSs of original Mw, high Mw, medium Mw and low Mw gave an encapsulation of 70.27,68.37,64.01 and 64.48%, respectively, but showed no influencing on the Vit.D3 encapsulation efficiency.On the CS-TPP-Ca- Vit.D3 nanoparticle size, it changed in a range of 360~580 nm as using Vit.D3/Ca concentration ratio from 0.5/100 to 4/800μg/ mg, but decreased to 495 nm with the concentration ratio 5/1000μg/ mg. The particle size gradually decreased when CS Mw became lower (1100~500 nm) and insignificantly affected by CS/TPP mass ratio (502~546.6 nm). On the zeta potential of the nanoparticles, it was not irregular affected by the Vit.D3/Ca concentration ratio (+18.5~+22.5 mV), became lower with the increasing CS/TPP mass ratio (+28.1~+24.1 mV), and increased with the increasing CS/TPP mass ratio (+24.1~+34.6 mV ).Moreover, CS-TPP-Ca- Vit.D3 nanoparticle that were prepared with low CS Mw, CS/TPP mass ratio of 6/1 and Vit.D3/Ca concentration ratio of 5/1000μg/ mg released almost 0.8% Ca and 1% Vit.D3 in the simulated gastric and intestine system. |
