本次計畫內容如預期目標所示，提供(1)最佳化之微脂體 LPPC 製備方式及成分比例，(2)LPPC 基本性質之測試，如吸附蛋白質能力、吸附力穩定性測試、組成成份穩定性測試等，(3)LPPC 穩定吸附蛋白質之特性探討，如 ITC 及 pH test，(4)所吸附蛋白質之活性探討，分別量測含血 清培養液及不含血清培養液中之功能性測試，以上結果皆表示出本計畫所研究之 LPPC 其獨特 性及新穎性。
基於此計畫所開發之微脂體特性，未來於犬類疫苗之開發，如包覆藥物並吸附導向分子以 治療腫瘤，甚至是人類疫苗方面皆可有效利用。其製備時間短且產量大，再加上其穩定吸附之 特性，可以避免未完全之化學反應對活體之傷害及所結合蛋白活性之喪失，因此在生醫產業之 應用性將非常地廣，甚至在生物材料及生物偵測實驗中皆可有效利用。
感謝國科會提供之研究經費，本計畫中之研究已撰寫並準備投稿至國際知名期刊，而其特 性分別在台灣及美國兩地申請專利中(專利申請程序進行中)。
出國報告（出國類別： C 類國際會議）
題目：International Conference on Materials for Advanced Technology 2009
服務機關：生化工程研究所
姓名職稱：廖光文 副教授
前往國家：新加坡 Suntech
出國期間：2009/06/28~07/03
報告日期：2009/06/30
參與國際型研討會 (ICMAT 2009)
出國報告書
98 年 7 月 10 日
報告內容：
一、參加經過
6/28 Day1 出發至新加坡，到研討會會場(Suntech city mall)報到和拿取相關資料 6/29 Day2 參與大會邀請學者演講
6/30 Day3 海報展覽及口頭講說 7/01 Day4 參與大會邀請學者演講 7/02 Day5 參與大會邀請學者演講
7/03 Day6 參與大會邀請學者演講，返國
二、心得（可含照片）
建立實驗室至今以發展腫瘤免疫治療方向努力，研究領域包含胃幽門螺旋桿菌之熱休克 蛋白致病機制和利用微脂體複合體於免疫治療平台開發，而對於微脂體在生醫材料的應 用發展已有初步研究成果，因此選擇在新加坡舉辦生醫材料相關技術的研討會，此次研 討會之行，除了分享目前實驗室的研究成果外，也藉此了解國際間於生醫材料上的發展。
參與研討會邀請的學者演講這幾天中，其中有學者利用 DNA 組成不同的結構，建立新 穎的傳送平台，擁有專一性且生物可分解性之特性，有趣的是學者利用此平台搭配 hydrogel 於生物體外製造蛋白質，節省時間及增加效率，有別於傳統式利用細菌或是細 胞，利用此新穎方法，激發我和學生們需要利用新方向去思考自我身旁之資源，而另外 有學者是利用親水疏水性質不同之聚合物合成複合體成功增加藥物在免疫治療上的療 效。 於海報展覽當天，和學生於會場分享本實驗室成果，研討會成員們對於本實驗室建 立的微脂體複合體的新穎吸附能力相關特性應用和搭配抗癌藥物之實驗成果，感到很有 興趣，且熱烈討論報告中有趣之發現和對於實驗結果表示肯定，此外比較其他國家實驗 室的成果分享，發現大多海報的展覽多著重於實驗目的和設計概念，但在實驗結果發表，
普遍都著重於建立生醫材料平台上基本特性的測試，並未有進一步應用之成果，不過本 實驗室開發之微脂體複合體此平台於生醫方面應用都較有卓越的成效及進展，突顯出交 通大學在生物科技領域發展之努力和進步，此外，研討會後感覺本實驗室和其他各國比 較仍具有一定之競爭力和潛力。
(a) 海報前合影之一(我與學生劉彥谷) (b) 海報前合影之二(我與學生陳家弘)
(c) 研討會報到地點 (d) 海報展覽區
(e) 會議演講廳外觀 (f) 演講廳會場
三、建議
a. 此次研討會之行，發現交通大學本校與其他學校於生物科技的競爭力並無相差太多，
但是在外語能力方面如聽力或演說較略顯不足，建議校方於外語能力的培養課程中可 以多著重於聽力和演說。
b. 建議贊助費用額度提高，因為與生物科技相關之比較大型國際型研討會大都集中於美 國、加拿大、德國等國，但除了該研討會註冊費較高外，餐旅費都相對來得比亞洲國 家更高。
四、攜回資料名稱及內容 a. 研討會摘要手冊 b. 研討會會議流程手冊 c. 研討會摘要內容光碟
五、其他 無
出國報告（出國類別： C 類國際會議）
題目：International Conference on Materials for Advanced Technology 2009
服務機關：生化工程研究所
姓名職稱：劉彥谷 博班學生
前往國家：新加坡 Suntech
出國期間：2009/06/28~07/03
報告日期：2009/06/30
出國會議報告書
感謝國科會給予計畫經費之補助，使學生得以參加 2009 年 ICMAT & IUMRS-ICA (International Conference on Materials for Advanced Technologies 2009 & International Union of Materials Research Societies-International Conference in Asia 2009)，此次會議為第五屆 ICMAT 研討會，6/28~7/3 日於新加坡會議中心舉辦。經由國科會審核之計畫經費，使得學生能夠完 成 Development of Novel Liposome with Stable Protein Adsorption Ability for Bio-Application 之 研究內容，並於 2009/6/30 於會議之壁報中展示成果，會議中不乏各國研究學者前來詢問及討 論研究之內容，並在討論其中各國學者皆對此研究充滿高度興趣，甚至更深入的詢問研究之 做法及源起之動機，而學生亦在討論中發現此研究仍然充滿許多有趣的問題及題目可以深入 探討或應用，如可應用此可吸附蛋白質之微脂體吸附螢光蛋白及標靶性分子，以建構新型之 偵測試劑，並結合流式細胞儀將可快速且有效的分析細胞表面之標誌分子。
與會期間，學生亦與指導教授參與不少口頭報告會議，其中另人印象最為深刻的莫過於 曾發表於 Nature 之作者 Dan LUO，所報告之 DNA-based Hydrogels for Drug Delivery and P-gels for Protein Production without Any Living Cells，此篇作者利用 DNA 做為一種攜帶藥物進入到 細胞內部之載體，此構想源自於現今載體幾乎都是利用其帶電的特性吸附特定物質進而送入 至細胞內部，但往往這些載體都會具有些許的毒性，因此作者想利用細胞本身的物質來運送 藥物進入到細胞內部，此做法將可有效的降低載體對於細胞的相容性及毒性上的問題。如此 新穎的構想，的確令人眼睛為之一亮，並且提供藥物載體建構之另一種思維。但於壁報之展 示中，我們發現國內研究載體之內容相較起來較有新穎性，且提供之數據內容也較為完整，
因此，國科會所提供之載體相關之研究成果皆相當豐碩，但國外研究仍有我們該學習的地方，
譬如說其對照組較為完整，且使用之技術及儀器亦較為高級及方便，這些皆是我們國內研究 需要加強的地方。
出國報告（出國類別： C 類國際會議）
題目：International Conference on Materials for Advanced Technology 2009
服務機關：生化工程研究所
姓名職稱：陳家弘 碩班學生
前往國家：新加坡 Suntech
出國期間：2009/06/28~07/03
報告日期：2009/06/30
出國會議報告書
感謝老師和學姊給予我這個機會讓我出國學習，由此次準備新加坡之旅，學習如何從網 路上找尋適合領域的研討會，及事後註冊報名、辦理機票護照、訂購房間等等前置作業，到 後來於研討會會場中和學者們分享實驗結果，在討論切磋中了解自己本身實驗設計和分析結 果之優缺點，並且給我們一個很好的思考方向來改進。而且出國參加研討會對自己來說可以 了解其他領域或是相關領域的學者們的看法，也可以比較其他各國實驗室在生物科技的發 展，除此之外由研討會邀請的演講者吸收到許多新穎的想法，這樣的激盪下所激發出的科技 火花，讓自己更加成長茁壯。
此外英語的聽說能力對於這次研討會非常重要，因為在研討會中我們大多利用英語分享 及解說，常常在討論中會遇到口音不同或是使用詞彙不恰當，有時會有雞同鴨講的狀況發生，
所以我認為舉凡參加國際型的研討會都需要擁有，這樣在涉獵汲取新知外，和世界各國的先 進們都可以盡情的切磋討論。
綜合來說，參加這次新加坡研討會之旅，除了讓自己了解語言能力尚待加強外，也看到 其他相關領域學者的實驗架構想法和實驗設計進步，未來如果有機會的話，我會建議實驗室 同仁多多參加。
Development of Novel Liposome with Stable Protein encapsulation and delivery; they can entrap both hydrophilic and hydrophobic drugs. Liposome combined with targeting molecule, such as antibodies can deliver drug to specific site.
Therefore, retaining activities of targeting molecules or protein-drugs by convenient process would be an important issue to manufacture drug carriers. The novel liposome, Lipo-PEI-PEG C o m pl e x (L P P C ) , w a s c o m p o s e d o f l i p i d , polyethyleneimine and polyethylene glycol. With our procedure, TEM showed that polymers were dis per sed o n t he l ipi d l a yer of lip o so me.
Interestingly, LPPC could stably adsorb various proteins, such as antibodies, beta-glucuronidase etc., even 10-fold competitive protein could not replace pre-adsorbed proteins on LPPC, and adsorptive reaction was accomplishable within 20 min. The results showed that PEI played an important role in proteins adsorption and conspicuously different enthalpy pattern of LPPC was observed. Moreover, the proteins adsorbed on LPPC would retain their activities in serum contained medium. That is to say, LPPC could be applied to bio-application, such as adsorbed anti-VEGFR antibodies for specific target to cancer cell.
This kind of quick and stable adsorptive ability could improve medical material and vaccines.
Strategy
We developed a novel liposome-polymer complex, LPPC, which was a liposome modified by PEG and PEI without covalent conjugation. The LPPC was used to combine with proteins for investigating the bio-functions of proteins on the liposome.
Results
Figure.1. The microstructure of LPPC.
(A) Schematic diagram, (B) TEM and (C) SEM images showed the microstructure of LPPC, which had dense circle regions and hair-like surface.
A B C
100 nm
Substance Assay Method
Bovine serum albumin(BSA) Coomassie Plus Reagent FITC-conjugated BSA Spectrofluorometer Beta-glucuronidase ELISA
HRP-conjugated Antibody ELISA
Hp Hsp60 OD 280
Ureas B OD 280
DNA Transfection Table 1. Substances adsorbed with LPPC.
Figure 2. Protein adsorption ability of different liposome.
Liposome were manufactured with different components for investigating the effects on adsorptive abilities. Liposome could adsorb proteins when PEI was existed. (mean ± SD, n=6)
0.0
Total of Detected BSA (μg)
Figure 3. Adsorption capacity and timing test of LPPC (40 μg).
(A) LPPC was incubated with different amounts of BSA for 20min, and detected the quantities of BSA adsorbed on LPPC by ELISA. (B) LPPC was incubated with certain quantities of BSA-FITC for di fferent reacti on time, a nd a nal yze d by spectrofluorometer. (mean ± SD, n=6)
(A) (B)
Figure 4. Competition test of LPPC (40 μg) ( A) HRP-conj ugated antibodies, (B) beta-glucuronidases and (C) BSA-FITCs, which prior incubated with the LPPC, were competed by BSA.
The results showed that the pre-adsorbed proteins
NC
100 ddH0.2M Glycine Solution2O
Adsorbed BSA(%)
Figure 5. Effect of 0.2 M glycine solution to adsorptive ability.
LPPC was incubated with BSA before or after transferring into the 0.2 M glycine solution.
The results showed that the protein-adsorbed on LPPC would not be dissociated from the surface when it was transferred into the low pH glycine buffer (0.2 M); nevertheless, the low pH glycine buffer indeed lowered the interactions of protein and LPPC. (mean ± SD, n=6)
Figure 6.Activities of adsorbed proteins on LPPC in serum contained medium.
LPPC was incubated with beta-glucuronidase for 20mins. And the results showed that the beta-glucuronidase still existed on LPPC and retained their activities in serum contained DMEM. (mean ± SD, n=6)
Figure 7.Activities of adsorbed antibodies on LPPC.
LPPC was incubated with anti-CD3 and/or a nt i- CD28 monoc lonal a ntibodies f or stimulating cell proliferation and cytokine release of PBMC. The results revealed that the monoclonal antibodies could retained their activities to stimulate the cells. (mean ± SD, n=6)
1.LPPC could adsorb with several substance, such as proteins, antibodies and DNA, by PEI and this interaction might not be only
provided by electric force.
2.Pre-adsorbed proteins on LPPC could retain their activities, and would not be replaced by other proteins; thus, LPPC is a
Conclusion
Total of competing BSA (μg)
Pre-Adsorbed HRP-Ab (%)
Total of competing BSA (μg)
Pre-AdsorbedβG (%)
200 300 400 500 0 (PC) 0
50 100
Total of competing BSA (μg)
Pre-Adsorbed BSA-FITC (%)
A Novel Liposome Complex for Encapsulating Curcumin to Enhance the Antitumor Effect of Curcumin
College of Biological Science and Technology, National Chiao Tung University Yu-Ling Lin, Yen-Ku Liu, Chia-Hung Chen and Kuang-Wen Liao
T h e l i p o s o m e - b a s e d e n c a p s u l a t i o n o f chemotherapeutic drugs has been developed well for the increases in drug delivery and efficacy, and the decrease in side effect. Several natural extracts have been reported that they have the selective toxicities to n o r m a l a n d t u m o r c e l l s , H o w e v e r, t h e y a r e hydrophobic components and limited in their poor bioavailability. We firstly developed a novel cationic l i p o s o m e - P E G - P E I c o m p l e x ( L P P C ) w h i c h e n c a p s u l a t e d t h e h y d r o p h o b i c c u r c u m i n (curcumin/LPPC) and converted the curcumin to a v a i l a b l e h y d r o p h i l e . Tr a n s m i s s i o n e l e c t r o n microscopy image showed that the curcumin/LPPC had roughly spherical shape and hairlike surface. In addition, the curcumin/LPPC’s encapsulating rate of curcumin were 45-55% for different packaged formulae, and 90% of encased curcumin would stably release from curcumin/LPPC at 37℃ for 72 h. Surprisingly, the cytotoxic activities of the encapsulated curcumin were higher to 490% than free curcumin, even more 20-fold
T h e l i p o s o m e - b a s e d e n c a p s u l a t i o n o f chemotherapeutic drugs has been developed well for the increases in drug delivery and efficacy, and the decrease in side effect. Several natural extracts have been reported that they have the selective toxicities to n o r m a l a n d t u m o r c e l l s , H o w e v e r, t h e y a r e hydrophobic components and limited in their poor bioavailability. We firstly developed a novel cationic l i p o s o m e - P E G - P E I c o m p l e x ( L P P C ) w h i c h e n c a p s u l a t e d t h e h y d r o p h o b i c c u r c u m i n (curcumin/LPPC) and converted the curcumin to a v a i l a b l e h y d r o p h i l e . Tr a n s m i s s i o n e l e c t r o n microscopy image showed that the curcumin/LPPC had roughly spherical shape and hairlike surface. In addition, the curcumin/LPPC’s encapsulating rate of curcumin were 45-55% for different packaged formulae, and 90% of encased curcumin would stably release from curcumin/LPPC at 37℃ for 72 h. Surprisingly, the cytotoxic activities of the encapsulated curcumin were higher to 490% than free curcumin, even more 20-fold