201035311 . 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種藉由生物材料雞卵蛋白(OVA)及聚甘醇酸，乳酸/經 基乙酸共聚合物(PLGA)(poly D，L-lactide-co-glycolide)奈米複合物配方之調 整促進細胞效益’其目的：主要針對不同比例的聚亞乙胺(PEi/pDNA) (polyethylenimine)及結合雞卵蛋白（OVA)奈米複合物與聚亞乙胺 (PEI/pDNA)(polyethyIenimine)及結合聚甘醇酸，乳酸/經基乙酸共聚合物 (PLGA)(polyD ’ L-lactide-co-glycolide)奈米複合物配方進行分析，發現聚亞 ❹ 乙胺(PEI/pDNA)(polyethylenimine)及結合雞卵蛋白(〇VA)奈米複合物與聚 亞乙胺(PEI/pDNA)(polyethylenimine)及結合聚甘醇酸，乳酸/羥基乙酸共聚 合物(PLGA)(poly D，L-lactide-co-glycolide)奈米複合物配方之製劑會因聚亞 乙胺質量比(PEI/pDNAmass ratio)的比例而造成轉染效率的差異，和質體去 氧核糖核酸(DNA)比較其轉染率能提高到8至65倍，因此若能將聚亞乙胺 加上質體去氧核糖核酸及聚甘醇酸，乳酸/羥基乙酸共聚合物 (PEI/pDNA-PLGA)奈米複合物技術發展成轉染率高而毒性低之去氧核糖核 酸(DNA)疫苗載體’將可應用於藥物應用於藥物遞送傳輸系統及基因治療 【先前技術】 按，1997年香港禽流感爆發至今，肆虐亞洲十個國家和地區，造成家 禽養殖業引起巨大威脅與損失，直到目前為止禽流感還未受到有效的控 制，專豕擔心禽流感與流感病毒基因置換而產生超級禽流感病毒，造成全 球性傳染病；目前WHO (國際衛生組織）已建議禁止使用減毒活菌疫苗， 而去氧核糖核酸(DNA)疫苗較不具致病性，在製備及使用上較為安全，許多 學者已證明使用禽流感去氧核糖核酸(DNA)疫苗可具有免疫保護作用；但由 4 201035311 . 於質體去氧核糖核酸(dna)進入細胞的效率不佳，因此需要藉由藥物遞送技 術增進轉染的效率，來發揮去氧核糖核酸(DNA)疫苗功效；而常用的藥物遞 送系統如病毒載體的安全性及脂質體(丨ip〇S〇me)的成本過高等問題存在，故 可利用生物降解材料結合聚甘醇酸，乳酸/羥基乙酸共聚合物(PLGA)(po丨y D ’ L-lactide-co-glyC〇Hde)及雞印蛋白(OVA)來合成核酸疫苗物理性載體遞送 系統來改善； 近年來生物降解性之聚合物常被利用於藥物遞送系統，我們希望由乳 酸(lactic acid)及甘醇酸(giyC〇iic acid)縮合而成聚甘醇酸，乳酸/羥基乙酸共聚 〇 合物[p〇ly D ’ L]actide-c〇-glyc〇lide(PLGA)]具有良好的生物相容性 (biocompatibility) ’並結合廣泛用於食品蛋白的材料雞卵蛋白（chicken ovalbumin，OVA)和陽離子聚亞乙胺[(poly L如ine，pLL及 polyethylenimine，PEI)]，藉由調整不同比例之配方及反應時間，形成穩定 之奈米載體’所以我們為了有效篩選不同的配方，使用螢光蛋白基因φΕορρ) 質體為模式去氧核糖核酸(DNA) ’使用複合法製備不同配方的奈米載體，以 表面電荷和粒徑等物化性來分析，並以建立的細胞平台進行穿透促進試驗 及評估去氧核糖核酸(DNA)轉染效能，希望能發展高轉染性且低毒性的去氧 Q 核糖核酸(DNA)疫苗配方，可應用於藥物傳輸系統及基因治療的遞送載體 上0 【發明内容】 本發明係關於一種藉由生物材料雞卵蛋白(0VA)及聚甘醇酸，乳酸/羥 基乙酸共聚合物(PLGA ; poly D ’ L-lactide-co-glycolide)奈米複合物配方之 調整促進細胞效益，包含一螢光蛋白基因之質體(pEGFPplasmidEGFp)去氧 核糖核酸(DNA)萃取純化裝置、一質體去氧核糖核酸(DNA)載體複合物之製 備裝置 '一質體去氧核糖核酸(DNA)複合物之物化性質評估裝置、一人類結 腸癌細胞株(CaCo-2)細胞平台建立装置細胞平台建立裝置、一流式細胞儀與 201035311 一細胞存活測試(ΜΤΤ)細胞毒性測試裝置所組成，係先將質體去氧核糖核酸 (DNA)透過螢光蛋白基因(pEGFp)質體去氧核糖核酸(DNA)萃取純化裝置進 行％取純化，將萃取純化後的質體去氧核糖核酸(DNA)藉有質體去氧核糖核 酸(DNA)載體複合物之製備裝置與生物相容性高(bi〇c〇mpatibUky)的聚甘醇 酸’乳酸/羥基乙酸共聚合物(PLGA)奈米複合物和雞即蛋白(〇VA奈米複合 物)結合螢光蛋白基因(pEGFP)質體去氧核糖核酸(DNA)，調整不同比例之配 方及反應時間來篩選不同配方的奈米載體，並透過質體去氧核糖核酸(DNA) 複合物之物化性質評估裝置所設置的測量表面電荷性質儀測定微粒表面 〇 (zeta)電位，測定後之微粒表面經由所設置之雷射粒徑分析儀測定粒徑分佈 情形，最後經由電子顯微鏡檢測完成物化性質評估，再透過人類結腸癌細 胞株(CaCo-2)細胞平台建立裝置與流式細胞儀進行穿透促進試驗及評估去 氧核糖核酸(DNA)轉染效能，發現聚亞乙胺加上質體去氧核糖核酸 (PEI/pDNA)及結合雞卵蛋白(ova)奈米複合物配方之製劑會因聚亞乙胺 (PEI)和質體去氧核糖核酸(pDNA)質量比(mass ratio)的比例而造成轉染效率 的差異，和質體去氧核糖核酸(DNA)比較其轉染率能提高到8至65倍，不 過於細胞存活測試(MTT)細胞毒性測試裝置中發現，殘留細胞轉染率仍維持 q 在55%至66%之間，符合聚亞乙胺(PEI)對細胞存在有毒性之前提，但市售 的細胞轉染(jetPEI-RGD)試劑殘留細胞轉染率則接近30% ,表示細胞轉染 (jetPEI-RGD)試劑雖然細胞轉染率，但細胞毒性也較高，因此若能將聚亞乙 胺加上質體去氧核糖核酸及聚甘醇酸，乳酸/經基乙酸共聚合物 (PEI/pDNA-PLGA)奈米複合物技術發展成轉染率高而毒性低之去氧核糖核 酸(DMA)疫苗載體’將可應用於藥物應用於藥物遞送傳輸系統及基因治療 【實施方式】 201035311 s玲參閱第一圖，為本發明糟由生物材料生物材料雞卵蛋白(〇va)及聚甘 醇酸’乳酸/羥基乙酸共聚合物(PLGA)(P〇ly D，L_lactide_co_giyc〇Hde)奈米 複合物配方之調整促進細胞效益之裝置結構方塊示意圖，由圖中可知，本 發明藉由生物材料雞卵蛋白(OVA)及聚甘醇酸，乳酸/羥基乙酸共聚合物 (PLGA)(poly D，L-lactide-co-glycolide)奈米複合物配方之調整促進細胞效 100，係包含一螢光蛋白基因(pEGFP)質體去氧核糖核酸(DNA)萃取純化裝 置1、一質體去氧核糖核酸(DNA)載體複合物之製備裝置2、一質體去氧核 糖核酸(DNA)載體複合物之物化性質評估裝置3、一人類結腸癌細胞株 〇 (CaCo_2)細胞平台建立裝置4、一流式細胞儀5與一檢測細胞存活率技術 (MTT)細胞毒性測試裝置6所組成，係先將質體去氧核糖核酸(DNA)透過蝥 光蛋白基因(pEGFP)質體去氧核糖核酸(DNA)萃取純化裝置1進行萃取純 化’再藉有質體去氧核糖核酸(DNA)載體複合物之製備裝置2將萃取純化質 體去氧核糖核酸(DNA)與生物相容性高(biocompatibility)的聚甘醇酸(PLGA) 奈米複合物和雞卵蛋白(OVA)奈米複合物進行配製成奈米載體，並透過質體 去氧核糖核酸(DNA)載體複合物之物化性質評估裝置3進行物化性評估，再 透過人類結腸癌細胞株(CaCo-2)細胞平台建立裝置4與流式細胞儀5進行穿 q 透促進試驗及評估去氧核糖核酸(DNA)轉染效能，最後透過檢測細胞存活率 技術(MTT)細胞毒性測試裝置6進行細胞毒性測試，完成一種藉由生物材料 雞卵蛋白(OVA)及聚甘醇酸’乳酸/經基乙酸共聚合物(pLGA)(poly D， L-lactide-co-glycolide)奈米複合物配方之調整促進細胞效益1〇〇。 請參閱第二圖’為本發明藉由生物材料雞卵蛋白(〇VA)及聚甘醇酸 (PLGA)奈米複合物配方之調整促進細胞效益之螢光蛋白基因(pEGFp)質體 去氧核糖核酸(DNA)萃取純化裝置方塊示意圖，由圖中可知，該螢光蛋白基 因(pEGFP)質體去氧核糖核酸(DNA)萃取純化裝置1，將帶有{蛋白基因 (EGFP)之基因片段螢光蛋白基因(pEGFP_C2，4.7kb，BD)的大腸桿菌}11以 1 : 100放大培養於細菌培養基（LB.Broth) 12，置於恆溫培養箱13中以37 201035311 C ’ 180rpm 培養 16 小時；利用 DNA 純化套組{(NudeoBond⑧PC2000kit (Macherey- Nagel Germany))}】4純化螢光蛋白基因(pEGFp)質體去氧核糖核 酸(DNA)，再以電泳15確認質體去氧核糖核酸(DNA)大小之動作。 請參閱第三圖，為本發明藉由生物材料雞卵蛋白(〇VA)及聚甘醇酸 (PLGA)奈米複合物配方之調整促進細胞效益之質體去氧核糖核酸(DNA)載 體複合物之製備裝置方塊示意圖，由圖可知：該質體去氧核糖核酸(DNA) 載體複合物之製備裝置2係將： 】·製備不同配方之聚甘醇酸+質體去氧核糖核酸(DNA)+雞卵蛋白 〇 (PLGA/pDNA/OVA)奈米複合物208 :將聚甘醇酸，乳酸/經基乙酸共 聚合物(PLGA)201及5仰螢光質體去氧核糖核酸(DNA)(pDNA)202分 別溶至150祕氯化納(NaCl)203溶液中，再將聚甘醇酸，乳酸/經基 乙酸共聚合物(PLGA)2 01溶液逐滴加至螢光質體去氧核糖核酸 (DNA)(pDNA)202溶液中震蘯(vortex)20秒’在室溫下靜置3〇分鐘， 即可配置成聚甘醇酸+質體(DNA)(PLGA/PDNA)204混合(complex) 質量比(mass ratio)分別為2:1; 4:1;若再加入5仲雞卵蛋白(〇va)2〇5 並震蘯(vortex)20秒後’在室溫下靜置30分鐘，即可配置成質體去氧 ❹ 核糖核酸(DNA)+聚甘醇酸+雞印蛋白(pDNA/PLGA/OVA)206混合 (complex)聚甘醇酸+質體去氧核糖核酸(DNA)(PLGA/pDNA)質量比 (mass ratio)為 2:1; 4:1);若再加入 5//g 支鏈聚亞乙胺(branch pei)207 展虚(vortex)20秒後’再室溫下靜置30分鐘，即可配置成聚亞乙胺(PEI) 表面修飾的質體去氧核糖核酸(DNA)+聚甘醇酸+雞印蛋白 (pDNA/PLGA/OVA)206 複合物； 2.製備不同配方之聚亞乙胺+質體去氧核糖核酸(!^八)+雞卵蛋白 (PEI/pDNA/OVA)232 奈米複合物： 分別將陽離子聚亞乙胺(PEI)209及質趙去氧核糖核酸(Dna) 230(5处)分別溶至丨οορ/βΟ/ηΜ的氣化鈉(NaCl)203溶液中，將聚亞乙 8 201035311 胺(PEI)聚合物231溶液逐滴加入到質體去氧核糖核酸(DNA)230溶液 震盪vortexlO秒後，在室溫下靜置30分鐘；加入不同體積(1〇，20〆) 之1吨/ml雞卵蛋白(OVA)205溶液雞卵蛋白+質體去氧核糖核酸 (DNA)(OVA/pDNA)質量比（massΓatio)為2:l;4:l)震盪(VOrtex)10 秒，在室溫下靜置30分鐘。 請參閱第四圖，為本發明藉由生物材料雞卵蛋白(OVA)及聚甘醇酸 (PLGA)奈米複合物配方之調整促進細胞效益之質體去氧核糖核酸(DNA)複 合物之物化性質評估裝置方塊示意圖，由圖中可知：該質體去氧核糖核酸 (DNA)複合物之物化性質評估裝置3係設有： 1. 測量表面電荷性質儀31表面電荷(zeta potential):評估經修飾後微粒 表面電位’研究中將已表面電荷比(zeta potential meter)測定微粒表面 (zeta)電位；取2哪微粒’加入3mZ之lwMCCI水溶液中，經超音波 水浴震盪1分鐘，在以表面電荷比(zeta potential meter)讀值； 2. 粒徑測定(以雷射粒徑分析儀32測定其粒徑分佈情形）： 分別稱不同配方之奈米複合物2mg，散佈於5 wi0.1 %聚山梨醇酯 80(Tween ’ P〇lyS〇rbate80)水溶液中；在以動力光散射粒徑分析儀測 定上述配製之懸浮物，即可得各分析物之粒徑分佈； 3. 電子顯微鏡33鏡檢： 將質體去氧核糖核酸(DNA)+雞卵蛋白+聚甘醇酸(pDNA-OVA-PLGA)奈米複合物之凍晶乾燥微奈米粒以固定液固定於碳膠鋁 盤上，再以臨界點乾燥機(critical point dryer，CPD，Hitachi HCP-2) 進行臨界點乾燥脫水，乾燥後的試片立即以導電膠帶及銀膠黏著於銘 台上(Aluminum slub) ’接著使用真空鍍金機{郎愈c〇ater(Bia卿 SC502，Fisons pic Registered 〇ffice，Englan叫做金覆膜(G〇ld palladium)處理’在電流60w下進行電鍍2分鐘，在樣本表面鍍上一 層導電層’完成樣品的前處理工作；使用掃描式電子顯微鏡(SEM)時， 201035311 電壓測定為5仟伏特(kV)來觀察表面型態及粒徑，並使用拍利得相紙 及掃描式電子顯微鏡(SEM)專用之黑白負片紀錄。 清蒼閱第五圖，為本發明藉由生物材料雞即蛋白(〇vA)及聚甘醇酸 (PLGA)奈米複合物配方之調整促進細胞效益之人類結騰癌細胞株(CaC〇_2) 細胞平台建立裝置方赫意圖，由圖巾可知，該人賴腸餘胞株吻㈤) 細胞平台建立裝置4，以人類結腸癌細胞株(CaC〇_2)細胞為人類大腸癌之細 胞株’以1%非必須氨基酸溶液4卜】〇%經加熱變性之胎牛血清溶液42、 青徽素G(benZy lepenicillin G)43⑽IU/wZ)及無血清的細胞培養基鍵黴素 〇 (streptomycin SUlfate)44〇00仰/w)配製成培養液，人類結腸癌細胞株 (Caco-2細胞)46加入培養液必，培養於幻它含空氣及5%二氧化碳的 培養中，培養後之細胞以含0.53wA/乙二胺四乙酸(£〇丁八)及〇 〇5%胰蛋白酶 (trypsin)之水溶液處理10分鐘，加入細胞培養基{Duibecc〇，s M〇dified Eagles’Mediimi(DMEM) }47，在以每平方公分5χ1〇6個細胞的密度種植於六 孔培養盤48(6-wellPlate) ’加入培養液後在培養箱中培養一週，以達到細胞 已佔滿生長平面空間(confluence)。 請參閱第六圖’為本發明藉由生物材料OVA及PLGA奈米複合物配方 Q 之藉由流式細胞儀評估不同配方之聚亞乙胺+質體去氧核糖核酸(DNA)+雞 卵蛋白+聚甘醇酸PEI/pDNA/OVA/PLGA奈米複合物的轉染效率方塊示意 圖，由圖中可知’該流式細胞儀5 ’於六孔培養盤48之每一孔中種植約8x 1〇5株細胞’以含有10%血清的細胞培養基(FBS/DMEM)51培育48小時， 分別將下列不同的配方與無血清的細胞培養基(serum-free/DMEM). 52滿合 後加入六孔培養盤孔48中；在37°C ’ 5%C〇2的培養箱中培養24小時後， 將培養基(medium)換成含有20%FBS之DMEM ’繼續培養直到48小時； 以倒立式榮光顯微鏡53觀察計數具有綠螢光蛋白產生之細胞個數，將每個 細胞培養皿(culture dish)中培養的細胞，以細胞刮刀連同培養液到下細胞， 以3，OOO(rpm)離心10分鐘收集細胞，再以磷酸緩衝溶液(PBS)清洗兩次， 201035311 去除磷酸緩衝溶液(PBS)後，以最終濃度70%冰酒精於4°C固定細胞；在以 3,000(rpm)離心10分鐘1將細胞重新懸浮於磷酸緩衝溶液(PBS)，加入0.5/"/ 含有;ε典化丙σ定溶液(Propidium iodide)(PI ’ 50 g/w/)的墙酸緩衝溶液(PBS)緩衝 液及100 /ig/m/核耱核酸(RnasA)於37 °C下處理1小時（最終濃度 0.4/^/m/)，輕緩震動離心管使細胞均勻懸浮，將離心管在室溫中避光4〇 分鐘’之後將細胞懸浮液移至康氏管中，利用（Beckman)公司的(Epics-ALTRA)流式細胞儀分析收集儀進行流式細胞分析(jj〇w anaiySis)。 請參閱第七圖’為本發明藉由生物材料雞卵蛋白(〇VA)及聚甘醇酸 O (PLGA)奈米複合物配方之檢測細胞存活率技術(MTT)細胞毒性測試裝置方 塊不意圖，由圖中可知，該檢測細胞存活率技術(Μττ)細胞毒性測試裝置 ό ’將配好的檢測細胞存活率技術(Μχτ)61加入96_培養皿(96_wenplate) 62(5000cell/well)中，置於培養箱中（1〜2hr.)，吸掉檢測細胞存活率技術 (MTT)61溶液並加入二甲基亞楓(DMS〇)63，置於震盪器(TheBellyDancer) 64上搖晃1〇〜20min，最後利用(MRXTC)酵素活性分析儀(ELISA)65以 57〇nm波長測得吸光值。 上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例 〇 麟肋關本發明之專利顧，絲麟本發明技髓神所為之等效實 施或變更’均應包含於本案之專利範圍中。 綜上所述，本案不但在技術思想上確屬創新’並能較習用物品增進上 述夕項功效，應已充分符合新穎性及進步性之法定發明專利要件，爰依法 提出申請，騎貴局核准本件發明專利申請案，以勵發明，至感德便。 【圖式簡單說明】 第-圖為本發明藉由生物材料雞印蛋白(〇VA)及聚甘醇酸，乳酸/經基乙酸 共聚合物(PLGA)㈣y D ’ L-〗actide-CO俞dide)奈米複合物配方之調 201035311 整促進細胞效益之裝置結構方塊示意圖。 第二圖為本發明藉由生物材料雞卵蛋白(OVA)及聚甘醇g^PLGA)奈米複 合物配方之調整促進細胞效益之螢光蛋白基因(pEGFP)質體去氧核 糖核酸(DNA)萃取純化裝置方塊示意圖。 弟二圖為本發明精由生物材料難即蛋白(OVA)及聚甘醇酸(PLGA)奈米複合 物配方之調整促進細胞效益之質體去氧核糖核酸(DNA)載體複合物 之製備裝置方塊示意圖。 第四圖為本發明藉由生物材料雞卵蛋白(OVA)及聚甘醇酸(PLGA)奈米複合 Q 物配方之調整促進細胞效益之質體去氧核糖核酸(DNA)複合物之物 化性質評估裝置方塊示意圖。 第五圖為本發明藉由生物材料雞卵蛋白(OVA)及聚甘醇酸(PLGA)奈米複合 物配方之調整促進細胞效益之人類結腸癌細胞株(CaCo-2)細胞平台 建立裝置方塊示意圖。 第六圖為本發明藉由生物材料雞卵蛋白(OVA)及聚甘醇酸(PLGA)奈米複合 物配方之藉由流式細胞儀評估不同配方之聚亞乙胺+質體去氧核糖 核酸(DNA)+雞卵蛋白+聚甘醇酸PEI/pDNA/OVA/PLGA奈米複合物 ^ 的轉染效率方塊示意圖。 第七圖為本發明藉由生物材料雞卵蛋白(OVA)及聚甘醇酸(PLGA)奈米複合 物配方之檢測細胞存活率技術(MTT)細胞毒性測試裝置方塊示意圖。 【主要元件符號說明】 1…螢光蛋白基因(pEGFP)質體去氧核糖核酸(DNA)萃取純化裝置 11…蛋白基因(EGFP)基因片段之大腸桿菌 12."細菌培養基(LB.Broth) 13…恆溫培養箱 14...DNA 純化套組(NucleoBond®PC2000 kit) 201035311 15...電泳 2·..質體去氧核糖核酸(DNA)載體複合物之製備裝置 201…聚甘醇酸，乳酸/羥基乙酸共聚合物(pLGA) 202…質體去氧核糖核酸(DNA)(pDNA) 203…氯化鈉(NaCl) 204…聚甘醇酸+質體DNA(PLGA/pDNA) 205…雞即蛋白(〇VA) 206…質體去氧核糖核酸(DNA)+聚甘醇酸+雞卵蛋白(pDNA/PLGA/OVA) 207v •支鏈聚亞乙胺(branch PEI) 208…聚甘醇酸+質體去氧核糖核酸(DNA)+雞卵蛋白(pDNA/PLGA/OVA)奈 米複合物 209…陽離子聚亞乙胺(PEI) 230··.去氧核糖核酸(DNA) 231…聚亞乙胺(PEI)聚合物 232…聚亞乙胺+質體去氧核糖核酸(DNA)+雞卵蛋白（PEI/pDNA/OVA) 3.. .質體去氧核糖核酸(DNA)載體複合物之物化性質評估裝置 3】…測量表面電荷性質儀 32…雷射粒徑分析儀 33…電子顯微鏡 4.. .人類結腸癌細胞株(CaCo-2)細胞平台建立裝置 41…非必須氨基酸溶液 42.. .加熱變性之胎牛血清溶液 43…青黴素 G(benzy lepenicillin G) 44 …鏈黴素(Streptomycin) 45…培養液 46…人類結腸癌細胞株(CaCo-2)細胞 13 201035311 47…細胞培養基(DMEM) 48.. .六孔培養盤 5.. .流式細胞儀 51…含血清的細胞培養基(FBS/DMEM) 52…無血清的細胞培養基(serum-free/DMEM) 53.. .倒立式螢光顯微鏡 6·.·檢測細胞存活率技術(MTT)細胞毒性測試裝置 61.. .檢測細胞存活率技術(MTT) ❹ 62...96-培養皿(96-well plate) 63.. .二甲基亞楓(DMSO) 64 …震遭器(The Belly Dancer) 65…酵素活性分析儀(MRXTC) 100…藉由生物材料雞卵蛋白(OVA)及聚甘醇酸，乳酸/羥基乙酸共聚合物 (PLGA)(poly D，L-lactide-co-glycolide)奈米複合物配方之調整促進細胞 效益 〇 14201035311 . VI. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a biomaterial chicken egg protein (OVA) and polyglycolic acid, lactic acid/glycolic acid copolymer (PLGA) (poly D, L-lactide-co-glycolide) Nanocomposite formulation adjustment promotes cell efficiency' Purpose: Mainly for different proportions of polyethylenimine (PEi/pDNA) (polyethylenimine) and combined with egg albumin (OVA) nanocomposite Analysis with polyethylidene (PEI/pDNA) (polyethyIenimine) and polyglycolic acid, lactic acid/polyacetate-based copolymer (PLGA) (polyD ' L-lactide-co-glycolide) nanocomposite , found that polyethylenimine (PEI / pDNA) (polyethylenimine) and combined chicken egg protein (〇VA) nanocomposite and polyethylamine (PEI / pDNA) (polyethylenimine) and combined polyglycolic acid, lactic acid / The formulation of the glycolic acid copolymer (PLGA) (poly D, L-lactide-co-glycolide) nanocomposite will result in a difference in transfection efficiency due to the ratio of PEI/pDNA mass ratio. Compared with plastid deoxyribonucleic acid (DNA), the transfection rate can be increased by 8 to 65 times. If polyethyliamine can be added to plastid deoxyribonucleic acid and polyglycolic acid, the lactic acid/glycolic acid copolymer (PEI/pDNA-PLGA) nanocomposite technology can be developed to have high transfection efficiency and low toxicity. Oxygen ribonucleic acid (DNA) vaccine vector will be applied to drugs for drug delivery and transmission systems and gene therapy. [Previous technology] According to the outbreak of avian flu in Hong Kong in 1997, it has ravaged ten countries and regions in Asia, causing poultry farming. Huge threats and losses, so far the bird flu has not been effectively controlled, specifically worrying about the avian flu and influenza virus gene replacement to produce avian flu virus, causing global infectious diseases; currently WHO (International Health Organization) has recommended prohibition The use of live attenuated vaccines, while DNA vaccines are less pathogenic and safer to prepare and use, many scholars have demonstrated that the use of avian influenza DNA (DNA) vaccines can have immunoprotective effects. But by 4 201035311 . The efficiency of plastid deoxyribonucleic acid (dna) entering cells is poor, so it is necessary to improve the efficiency of transfection by drug delivery technology. To play the role of DNA vaccine; the safety of commonly used drug delivery systems such as viral carriers and the high cost of liposomes (丨ip〇S〇me) exist, so biodegradable materials can be combined. Polyglycolic acid, lactic acid/glycolic acid copolymer (PLGA) (po丨y D 'L-lactide-co-glyC〇Hde) and chicken printed protein (OVA) to synthesize a nucleic acid vaccine physical carrier delivery system to improve; In recent years, biodegradable polymers are often used in drug delivery systems. We hope to condense lactic acid and glycolic acid (giyC〇iic acid) to form polyglycolic acid, lactic acid/glycolic acid copolymer complex. [p〇ly D 'L]actide-c〇-glyc〇lide (PLGA)] has good biocompatibility ' combined with the widely used material for food proteins, chicken ovalbumin (OVA) and Cationic polyethylamine [(poly L such as ine, pLL and polyethylenimine, PEI)], by adjusting different ratios of formula and reaction time to form a stable nano carrier' so we use fluorescent to effectively screen different formulations Protein gene φΕορρ) The plastid is a model of deoxyribonucleic acid (DNA). The nanocarriers of different formulations were prepared by a composite method, and analyzed by physicochemical properties such as surface charge and particle size, and the penetration promotion test and evaluation of deoxygenation were performed using the established cell platform. Ribonucleic acid (DNA) transfection efficiency, hope to develop a highly transfectible and low toxicity deoxygenation of ribonucleic acid (DNA) vaccine, which can be applied to drug delivery systems and gene therapy delivery vehicles. The invention relates to an adjustment promoting cell by a biomaterial chicken egg protein (0VA) and a polyglycolic acid, a lactic acid/glycolic acid copolymer (PLGA; poly D 'L-lactide-co-glycolide) nanocomposite formulation Benefits, including a fluorescent protein gene plastid (pEGFPplasmidEGFp) deoxyribonucleic acid (DNA) extraction and purification device, a plastid deoxyribonucleic acid (DNA) carrier complex preparation device 'a plastid deoxyribonucleic acid ( Physicochemical property evaluation device for DNA) complex, a human colon cancer cell line (CaCo-2) cell platform establishment device cell platform establishment device, first-class cytometer and 201035311 one cell survival test (ΜΤΤ) The toxicity test device is composed of a plastid deoxyribonucleic acid (DNA) through a fluorescent protein gene (pEGFp) plastid deoxyribonucleic acid (DNA) extraction and purification device for purification, and the purified plastid is extracted. Deoxyribonucleic acid (DNA) preparation device with plastid deoxyribonucleic acid (DNA) carrier complex and biocompatible (bi〇c〇mpatibUky) polyglycolic acid 'lactic acid/glycolic acid copolymer (PLGA) nanocomposite and chicken protein (〇VA nanocomposite) bind fluorescent protein gene (pEGFP) plastid deoxyribonucleic acid (DNA), adjust the formulation and reaction time of different ratios to screen different formulations a nanocarrier, and measuring the surface zeta potential of the microparticle by a measuring surface charge property set by a physicochemical property evaluation device of the plastid deoxyribonucleic acid (DNA) complex, and measuring the surface of the microparticle via the set laser The particle size analyzer measures the particle size distribution, and finally evaluates the physicochemical properties through electron microscopy, and then passes through the human colon cancer cell line (CaCo-2) cell platform establishment device and flow cytometry for penetration promotion test and evaluation. Deoxyribonucleic acid (DNA) transfection efficiency, found that polyethylidene plus plastid deoxyribonucleic acid (PEI / pDNA) and a combination of chicken egg protein (ova) nanocomposite formulation will be due to polyethylamine ( The difference in transfection efficiency between PEI) and plastid deoxyribonucleic acid (pDNA) mass ratio can increase the transfection rate by 8 to 65 times compared with plastid deoxyribonucleic acid (DNA). However, in the cell viability test (MTT) cytotoxicity test device, it was found that the residual cell transfection rate was maintained at between 55% and 66%, which was consistent with the toxicity of polyethylamine (PEI) to the cells, but Commercial cell transfection (jetPEI-RGD) reagent residual cell transfection rate is close to 30%, indicating that cell transfection (jetPEI-RGD) reagent, although cell transfection rate, but also high cytotoxicity, so if it can be polymerized Ethylamine plus plastid deoxyribonucleic acid and polyglycolic acid, lactic acid/transacetic acid-based copolymer (PEI/pDNA-PLGA) nanocomplex technology developed into a high transfection rate with low toxicity of deoxyribonucleic acid (DMA) vaccine vector 'will be applied to drugs for drug delivery and delivery systems and gene therapy [implementer] 】 201035311 s Ling see the first picture, the invention is the biological material biomaterial chicken egg protein (〇va) and polyglycolic acid 'lactic acid / glycolic acid copolymer (PLGA) (P〇ly D, L_lactide_co_giyc〇Hde The structure of the nanocomposite formulation is adjusted to promote cell efficiency. The structure of the device is shown in the figure. The present invention utilizes the biomaterial chicken egg protein (OVA) and polyglycolic acid, lactic acid/glycolic acid copolymer (PLGA). (poly D, L-lactide-co-glycolide) nano-complex formulation to promote cell cytokines 100, including a fluorescent protein gene (pEGFP) plastid deoxyribonucleic acid (DNA) extraction and purification device 1, a quality Preparation device of bulk deoxyribonucleic acid (DNA) carrier complex 2, physicochemical property evaluation device of monoplast deoxyribonucleic acid (DNA) carrier complex 3, a human colon cancer cell line (CaCo_2) cell platform establishing device 4. First-class cytometer 5 and a cell viability testing technology (MTT) cytotoxicity test device 6 consisting of plastid deoxyribonucleic acid (DNA) through the sputum protein gene (pEGFP) plastid deoxyribose Nucleic acid (DNA) extraction and purification device 1 Extraction and purification of plastid deoxyribonucleic acid (DNA) and biocompatibility of polyglycolic acid (PLGA) by preparative device 2 using purified plastid deoxyribonucleic acid (DNA) carrier complex The nanocomposite and the chicken egg protein (OVA) nanocomposite are formulated into a nanocarrier, and the physicochemical property evaluation device 3 of the plastid deoxyribonucleic acid (DNA) carrier complex is used for physicochemical evaluation, and then Through the human colon cancer cell line (CaCo-2) cell platform establishment device 4 and flow cytometry 5 to carry out the penetration promotion test and evaluate the efficiency of DNA transfection, and finally pass the detection cell survival rate technology (MTT) The cytotoxicity test device 6 performs a cytotoxicity test to complete a biomaterial chicken egg protein (OVA) and a polyglycolic acid 'lactic acid/transacetic acid copolymer (pLGA) (poly D, L-lactide-co-) Glycolide) The adjustment of the nanocomposite formula promotes cell benefits. Please refer to the second figure 'The fluorescence-promoting gene (pEGFp) plastid deoxygenation that promotes cell efficiency by adjusting the biomaterial chicken egg protein (〇VA) and polyglycolic acid (PLGA) nanocomposite formulations. A schematic diagram of a ribonucleic acid (DNA) extraction and purification device. As shown in the figure, the fluorescent protein gene (pEGFP) plastid deoxyribonucleic acid (DNA) extraction and purification device 1 will carry a gene fragment of {protein gene (EGFP). The fluorescent protein gene (pEGFP_C2, 4.7 kb, BD) of Escherichia coli}11 was cultured in a bacterial medium (LB. Broth) 12 at a magnification of 1:100, and placed in a constant temperature incubator 13 and cultured at 37 201035311 C '180 rpm for 16 hours; Purification of the fluorescent protein gene (pEGFp) plastid deoxyribonucleic acid (DNA) using the DNA purification kit {(NudeoBond8PC2000kit (Macherey- Nagel Germany))} 4, and confirming the plastid deoxyribonucleic acid (DNA) by electrophoresis 15 The action of size. Please refer to the third figure, which is a plastid deoxyribonucleic acid (DNA) carrier complex which promotes cell efficiency by adjusting the formulation of biomaterial chicken egg protein (〇VA) and polyglycolic acid (PLGA) nanocomposite. Block diagram of the preparation device of the object, it can be seen from the figure that the preparation device 2 of the plastid deoxyribonucleic acid (DNA) carrier complex will: prepare a different formula of polyglycolic acid + plastid deoxyribonucleic acid (DNA) + chicken egg yolk (PLGA/pDNA/OVA) nanocomposite 208: polyglycolic acid, lactic acid/transacetic acid copolymer (PLGA) 201 and 5 fluorescein deoxyribonucleic acid (DNA) (pDNA) 202 is dissolved in 150 sodium chloride (NaCl) 203 solution, and then polyglycolic acid, lactic acid / transacetic acid copolymer (PLGA) 2 01 solution is added dropwise to the fluorescent body Oxygen ribonucleic acid (DNA) (pDNA) 202 solution vortex for 20 seconds 'Standing at room temperature for 3 ,, can be configured as polyglycolic acid + plastid (DNA) (PLGA / PDNA) 204 The mass ratio is 2:1; 4:1; if 5 broiler egg protein (〇va) 2〇5 is added and vortex is shaken for 20 seconds, 'restore at room temperature Set for 30 minutes, you can configure it to Plastid deoxyribonucleic acid (DNA) + polyglycolic acid + chicken-printed protein (pDNA/PLGA/OVA) 206 complex polyglycolic acid + plastid deoxyribonucleic acid (DNA) (PLGA/pDNA) The mass ratio is 2:1; 4:1); if 5//g branched polyethylene pei 207 is added, vortex is 20 seconds later, and then left at room temperature for 30 In minutes, it can be configured as a polyethylideneamine (PEI) surface-modified plastid deoxyribonucleic acid (DNA) + polyglycolic acid + chicken-printed protein (pDNA/PLGA/OVA) 206 complex; 2. Preparation of different formulations Polyethylamine + plastid deoxyribonucleic acid (!^8) + chicken egg protein (PEI/pDNA/OVA) 232 nanocomposite: cationic polyethylamine (PEI) 209 and deoxygenated Ribonucleic acid (Dna) 230 (5) was dissolved in a solution of sodium oxalate (NaCl) 203 of 丨οορ/βΟ/ηΜ, and a solution of poly(A) 8 201035311 amine (PEI) polymer 231 was added dropwise to the plastid. Deoxyribonucleic acid (DNA) 230 solution was shaken for vortexlO seconds, and then allowed to stand at room temperature for 30 minutes; different volumes (1 〇, 20 〆) of 1 ton / ml chicken egg protein (OVA) 205 solution chicken egg protein + The plastid deoxyribonucleic acid (DNA) (OVA/pDNA) mass ratio (mass Γatio) is 2:1; 4: l) Vortex (VOrtex) for 10 seconds and let stand for 30 minutes at room temperature. Please refer to the fourth figure, which is a plastid deoxyribonucleic acid (DNA) complex which promotes cell efficiency by adjusting the formulation of biomaterial chicken egg protein (OVA) and polyglycolic acid (PLGA) nanocomposite. The block diagram of the physicochemical property evaluation device is shown in the figure: The physicochemical property evaluation device 3 of the plastid deoxyribonucleic acid (DNA) complex is provided: 1. Measuring the surface charge property meter 31 zeta potential: evaluation After modification, the surface potential of the microparticles will be measured by the zeta potential meter. The 2 microparticles were added to a 3 mZ lwMCCI aqueous solution and shaken for 1 minute in an ultrasonic bath. Charge ratio (zeta potential meter) reading; 2. Particle size measurement (measured by laser particle size analyzer 32): 2mg of different formulations of nanocomposite, scattered in 5 wi0.1% In the aqueous solution of polysorbate 80 (Tween 'P〇lyS〇rbate 80); the particle size distribution of each analyte can be obtained by measuring the suspended matter prepared by a dynamic light scattering particle size analyzer; 3. Electron microscope 33 mirror Inspection: plastid Freeze-dried micro-nanoparticles of deoxyribonucleic acid (DNA) + chicken egg protein + polyglycolic acid (pDNA-OVA-PLGA) nanocomposite are fixed on a carbon offset aluminum plate with a fixative solution and dried at a critical point. The critical point dryer (CPD, Hitachi HCP-2) was subjected to critical point drying and dehydration. The dried test piece was immediately adhered to the aluminum plate by conductive tape and silver glue. Then vacuum vacuum plating machine was used. 〇ater (Biaqing SC502, Fisons pic Registered 〇ffice, Englan called G〇ld palladium treatment 'electroplating at 60w for 2 minutes, plating a layer of conductive layer on the surface of the sample' to complete the pre-treatment of the sample When using a scanning electron microscope (SEM), the voltage of 201035311 was measured at 5 volts (kV) to observe the surface morphology and particle size, and a black and white negative film record for Polaroid photographic paper and scanning electron microscope (SEM) was used. Qing Cang read the fifth figure, which is a human forcing cancer cell line (CaC〇_) which promotes cell efficiency by adjusting the formulation of biomaterial chicken protein (〇vA) and polyglycolic acid (PLGA) nanocomposite. 2) Cell platform establishment device Fanghe intention, The towel can be seen, the person's residual cell strain (5)) cell platform establishment device 4, human colon cancer cell line (CaC〇 2) cells for human colorectal cancer cell line '1% non-essential amino acid solution 4 Bu 〇% is prepared by heat-denatured fetal bovine serum solution 42, benzyl lepenicillin G 43 (10) IU/wZ) and serum-free cell culture medium streptomycin SUlfate 44〇00 //w) The culture medium, human colon cancer cell line (Caco-2 cell) 46 is added to the culture medium, and cultured in a medium containing air and 5% carbon dioxide, and the cultured cells are contained with 0.53 wA/ethylenediaminetetraacetic acid (£ The solution was treated with a solution of 5% 5% trypsin for 10 minutes, and added to the cell culture medium {Duibecc〇, s M〇dified Eagles' Mediiimi (DMEM) } 47 at 5χ1〇6 cells per square centimeter. The density was planted in a six-well plate 48 (6-wellPlate). After the medium was added, it was cultured for one week in an incubator to achieve a cell-filled growth confluence. Please refer to the sixth figure for the evaluation of different formulations of polyethylamine + plastid deoxyribonucleic acid (DNA) + chicken eggs by flow cytometry using biomaterial OVA and PLGA nanocomposite formulation Q. Block diagram of transfection efficiency of protein + polyglycolic acid PEI/pDNA/OVA/PLGA nanocomposite, as shown in the figure, 'the flow cytometer 5' is planted in each well of a six-well culture plate 48 about 8x 1〇5 cells were incubated with cell culture medium (FBS/DMEM) 51 containing 10% serum for 48 hours, and the following different formulations were mixed with serum-free cell culture medium (serum-free/DMEM). The wells were cultured in wells 48; after 24 hours of incubation in a 37 ° C '5% C 2 incubator, the medium was replaced with DMEM containing 20% FBS and culture was continued until 48 hours; The microscope 53 observes and counts the number of cells having green fluorescent protein production, and the cells cultured in each cell culture dish are centrifuged at 3,000 (rpm) for 10 minutes with a cell scraper and a culture solution to the lower cells. Collect the cells and wash them twice with phosphate buffer solution (PBS). After flushing the solution (PBS), fix the cells at 4 ° C with a final concentration of 70% iced alcohol; resuspend the cells in phosphate buffer solution (PBS) by centrifugation at 3,000 (rpm) for 10 minutes. Add 0.5/"/ ; ypical solution of Propidium iodide (PI ' 50 g/w/) in wall acid buffer solution (PBS) buffer and 100 /ig/m/nuclear nucleic acid (RnasA) at 37 °C 1 hour (final concentration 0.4 / ^ / m /), gently shake the centrifuge tube to evenly suspend the cells, the tube is protected from light for 4 minutes at room temperature ' after the cell suspension is moved to the Conduit tube, use ( Beckman)'s (Epics-ALTRA) flow cytometry analyzer collects flow cytometry (jj〇w anaiySis). Please refer to the seventh figure' for the present invention. The cell viability test (MTT) cytotoxicity test device is not intended by the biomaterial chicken egg protein (〇VA) and polyglycolic acid O (PLGA) nanocomposite formulations. As can be seen from the figure, the detection cell survival rate technique (Μττ) cytotoxicity test device ό 'adds the prepared cell survival rate technique (Μχτ) 61 to 96_culture plate (96_wenplate) 62 (5000 cells/well). Place in the incubator (1~2hr.), aspirate the cell viability assay (MTT) 61 solution and add dimethyl sulfoxide (DMS 〇) 63, and shake it on the shaker (TheBellyDancer) 64~ At 20 min, the absorbance was finally measured using a (MRXTC) Enzyme Activity Analyzer (ELISA) 65 at a wavelength of 57 Å. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment of the present invention is based on the patent of the invention, and the equivalent implementation or modification of the invention of the invention is included in The patent scope of this case. In summary, this case is not only innovative in terms of technical thinking, but also can improve the above-mentioned effects of the above items in comparison with the customary items. It should fully comply with the statutory invention patent requirements of novelty and progressiveness, and apply for it according to law. This invention patent application, in order to invent invention, to the sense of virtue. [Simple diagram of the diagram] The first figure is the invention of the invention by the biomaterial chicken protein (〇VA) and polyglycolic acid, lactic acid / transacetic acid copolymer (PLGA) (four) y D 'L-〗actide-CO Yu Dide) nanocomposite formula adjustment 201035311 The whole structure of the device to promote cell efficiency. The second figure is a fluorescent protein gene (pEGFP) plastid deoxyribonucleic acid (DNA) that promotes cell efficiency by adjusting the formulation of biomaterial chicken egg protein (OVA) and polyglycol g(PL)) nanocomposite. ) Schematic diagram of the extraction and purification device. The second embodiment of the present invention is a preparation device for plastid deoxyribonucleic acid (DNA) carrier complex which is modified by the formulation of biomaterials (OVA) and polyglycolic acid (PLGA) nanocomposite to promote cell efficiency. Block diagram. The fourth figure shows the physicochemical properties of the plastid deoxyribonucleic acid (DNA) complex which promotes cell efficiency by adjusting the formulation of biomaterial chicken egg protein (OVA) and polyglycolic acid (PLGA) nanocomposite Q. A block diagram of the evaluation device. The fifth figure is a device for establishing a human colon cancer cell line (CaCo-2) cell platform for promoting cell efficiency by adjusting the formulation of biomaterial chicken egg protein (OVA) and polyglycolic acid (PLGA) nanocomposite. schematic diagram. The sixth figure shows the different formulations of polyethylamine + plastid deoxyribose by flow cytometry by the biomaterial chicken egg protein (OVA) and polyglycolic acid (PLGA) nanocomposite formulations. Schematic diagram of the transfection efficiency of nucleic acid (DNA) + chicken egg protein + polyglycolic acid PEI / pDNA / OVA / PLGA nanocomplex ^. Figure 7 is a block diagram showing the cell viability testing (MTT) cytotoxicity test device of the present invention by the biomaterial chicken egg protein (OVA) and polyglycolic acid (PLGA) nanocomposite formulations. [Explanation of main component symbols] 1... Fluorescent protein gene (pEGFP) plastid deoxyribonucleic acid (DNA) extraction and purification device 11... Protein gene (EGFP) gene fragment of Escherichia coli 12. " Bacterial culture medium (LB.Broth) 13...Immediate incubator 14...DNA purification kit (NucleoBond®PC2000 kit) 201035311 15... Electrophoresis 2·. Preparation of plastid deoxyribonucleic acid (DNA) carrier complex 201... Polyglycolic acid , lactic acid/glycolic acid copolymer (pLGA) 202... plastid deoxyribonucleic acid (DNA) (pDNA) 203... sodium chloride (NaCl) 204... polyglycolic acid + plastid DNA (PLGA/pDNA) 205... Chicken is protein (〇VA) 206... plastid deoxyribonucleic acid (DNA) + polyglycolic acid + chicken egg protein (pDNA / PLGA / OVA) 207v • branched polyethylamine (branch PEI) 208... polyglycan Alkyd + plastid deoxyribonucleic acid (DNA) + chicken egg protein (pDNA / PLGA / OVA) nanocomposite 209 ... cationic polyethylidene (PEI) 230 · · deoxyribonucleic acid (DNA) 231... Polyethylideneamine (PEI) polymer 232...polyethylamine + plastid deoxyribonucleic acid (DNA) + chicken egg protein (PEI/pDNA/OVA) 3.. plastid deoxyribonucleic acid (DNA) vector Composite material physicochemical property evaluation device 3 】...Measure surface charge property meter 32...Laser particle size analyzer 33...Electromicroscope 4. Human colon cancer cell line (CaCo-2) cell platform establishment device 41...Non-essential amino acid solution 42.. Heat denaturation Fetal bovine serum solution 43... penicillin G (benzy lepenicillin G) 44 ... streptomycin 45... culture solution 46... human colon cancer cell line (CaCo-2) cells 13 201035311 47... cell culture medium (DMEM) 48.. 6. Six-well culture plate 5.. Flow cytometer 51... Serum-containing cell culture medium (FBS/DMEM) 52... Serum-free cell culture medium (serum-free/DMEM) 53.. Inverted fluorescence microscope 6· .. Test Cell Survival Technology (MTT) Cytotoxicity Test Device 61.. Test Cell Survival Technology (MTT) ❹ 62...96 - Petri dish (96-well plate) 63.. . Dimethyl Yafeng (DMSO) 64 ... The Belly Dancer 65...Enzyme Activity Analyzer (MRXTC) 100...by biomaterial chicken egg albumin (OVA) and polyglycolic acid, lactic acid/glycolic acid copolymer (PLGA) (poly D, L-lactide-co-glycolide) nanocomposite formulation adjustment to promote cell efficiency 〇14