| 摘要: | 噴霧乾燥(spray-drying)已被用來發展生物科技上蛋白質生物製劑的製備與保存。噴霧乾燥的特色是它能同時快速保有顆粒形式和乾燥。因此，使用單一程序將水份從原來的水溶液中快速移除而得到固體顆粒的製劑產品引起生物製藥界的廣泛興趣。將陽電性高分子/DNA複合物以冷凍乾燥方式已證明可以穩定其DNA基因表現性達至少一年之久。然而使用快速簡單操作的噴霧乾燥方式來保存陽電性高分子/DNA複合物的研究卻極少。
本研究主要目的在探討噴霧乾燥處理後造成陽離子型高分子載體PEI/DNA複合物對基因表現的轉染效率，並評估藉由二類不同非緊縮形中性保護劑(如sucrose及polyethylene glycol; PEG 4000、10000、35000)的使用是否能夠有效的保護DNA在經過噴霧乾燥程序下維持DNA的安定性，以評估其發展成為長期安定製劑之可行性。
我們利用陽離子型高分子載體/DNA複合物polyethyleneimine (PEI)/DNA所形成的複合體(比例為1:1)來當作基因轉染的標準。蔗糖與PEG4000,10000,
35000在不同比例下(5,10,20,30％)與PEI/DNA形成二級的複合物。
Polymer/DNA複合體的形成中將PEI與DNA先分別的存在於水合的10% PEG中，再結合形成複合體；與將PEI/DNA先形成複合體後再存在於水合的10% PEG中。這些複合物接著馬上進行噴霧乾燥，所得到的產物進行基因轉染的研究，並測試細胞毒性與基因在瓊酯膠體上表現。
當赤裸的DNA分別與非緊縮形中性保護劑混合後再經過噴霧乾燥的處理，其質體DNA三级結構依舊大部份保持開環的形態。10％PEG與PEI/DNA(1：1)複合體能達到最高的產量。並且其轉染效果較佳以及在水中能有效的分解。我們選擇這混合比率爲了要做進一步的研究。
實驗之結果顯示，當PEI/DNA複合體與PEI/DNA複合體加入PEG4000進行轉染效率的分析:其轉染效率經過噴霧乾燥後就會消失。在PEI/DNA複合體加入PEG4000經過噴霧乾燥後亦沒有明顯的轉染效率。細胞的活性顯示這些製劑對細胞是沒有毒性；然而基因在瓊酯膠體的表現可以確定經過噴霧乾燥後PEI/DNA複合體即使加入帶高度負電荷的天門冬胺酸與正電荷PEI吸引，仍舊無法把赤裸DNA由PEI/DNA複合體中分離出來，故最後影響其轉染效率的表現。
結論為噴霧乾燥的過程是一種非常簡單的方法，可以保存多種的物質。不適合被用來當成一種保護劑，或者去保留它的生物活性。未來我們希望可以發現噴霧乾燥的過程尋找其他更好的保護劑。
Spray-drying method has been used for development of biotechnological protein preparation and preservation. The characteristic of spray-drying method is able to remain in particle form and keep dryness simultaneously. Using a single process to remove water from the original solution and obtain solid particle preparation attracts great interest by biopharmaceuticals. To preserve high molecular polycation/DNA complex by freeze-drying method has been identified and demonstrated to retain stability of DNA gene expression at least over one year. However, spray-drying method has not been extensively studied. The aims of this study are to investigate the factors that influence PEI/DNA transfection efficiency after spray-drying process and to evaluate protective efficiency of two different types of protective agents.
To investigation of transfection efficiency, polyethyleneimine(PEI)/DNA complex (the ratio is 1:1) was used as a criterion of gene transfection. Sucrose and PEG 4000, 10000, and 35000 were used as the protective agents in different ratios (5, 10, 20 and 30%) to form a secondary complex with PEI/DNA complex or become a mixture with DNA and PEI separately prior to make PEI/DNA complex. These complexes were then subsequently subjected to spray-drying process. The spray-drying products were then reconstituted and performed gene tranfection study. Their cytotoxicity and gene expression in agarose gel was also studied.
When naked plasmid DNA mixed with non-condense neutral protective agents after spray-drying process, most of DNA tertiary structure remained in open circular. Since 10％of all PEG mixed with PEI/DNA complex generated highest products, easily to be dissolved in water and possessed higher transfection efficiency, this mixing ratio was chosen for further studies. Results of transfection efficiency from fresh made PEI/DNA mixed with PEG 4000 was comparable to PEI/DNA complex. However, the efficiency was abolished after spray-drying process. Neither fresh prepared nor spray-drying prepared PEI/DNA mixed with other protective agents in either preparation methods show any significant transfection. Cell viability showed that all preparation were not-toxic to cells. Gene expressed in agarose gel also confirmed that protective agents could not be separated by polyaspartic acid from PEI/DNA complex after spray-drying process, subsequently affecting the transfection.
In conclusion, spray-drying process may be an easy method to perverse many types of material, it is not suitable for current chosen protective agents, in terms of reservation of their bioactivity. In the future work, we intend to find other better protective agents for this preserving process.Spray-drying method has been used for developm... |
