201103574 六、發明說明： 【發明所屬之技術領域】 jiili請案之弓丨用 本申請案主張美國臨時申請案61/055,784，申請曰為2〇〇8 年5月23日，之權益’其内容係完整地以引用方式納入本文 作為參考。 技術領垃 本發明廣義而言侧於藥物遞送，而更特別地係關於經皮 藥物遞送。 【先前技術】 經賴物遞送(TDD)可提供數種較包括注射及⑽遞送等 傳、’先技某方去更佳的優點。當相較於口服遞送時，可避免 胃腸的藥物代謝，減対渡效應(fct调effeet)，及提供藥物 之持續釋放多達七天。當她於帥時，可免除必要的疼 痛及感染的可紐。理論上，_之經皮賴可能在許多 治療性蛋”之遞送上有利，_蛋白f容級胃腸分解而呈 現較差的胃腸吸收。諸如干擾料蛋自細級血液清除，且 為了使其血錢度維持在高值，而需要以持續速率進行遞送， 美國專利案號 而且經皮魏裝置在㈣上·射容易 5,814,599) 〇 儘管具有這些優點，但因在由 一 —U為皮膚封於樂物之可穿透性低， 目前在臨床應用上僅非常少華 老物且尚無蛋白質或肽類以經皮 4 201103574 方式投藥。此低的可穿透性係由於角質層(sc)(其為皮膚最外 層，由被脂質雙層膜之充滿角質纖維之扁平 、死的細胞(角質細 胞)所組成）所致。脂質雙層臈之高度-規則排列構造，提供sc 不可牙透的特性（美國專利案號5,814,599)。 良方法。 【發明内容】 經皮藥物遞送提供可替代σ服遞送及注射之有利投藥方 式。然而皮膚職物之可穿雜相#低，故其應用僅限 於）數藥物。因此’迄今於該項技藝仍有需要提出解決前述缺 點及不適當性之方法，特別是結合一種用於經皮藥 改 良方法。 《队201103574 VI. Description of the invention: [Technical field to which the invention belongs] The application of the jiili case is based on the US provisional application 61/055, 784, and the application is the benefit of May 23, 2008. This is incorporated herein by reference in its entirety. Technical Summary The present invention broadly focuses on drug delivery, and more particularly on transdermal drug delivery. [Prior Art] By-Drug Delivery (TDD) can provide several advantages over injections and (10) delivery, etc. When compared to oral delivery, gastrointestinal drug metabolism, reduced efflux (fct teffet), and sustained release of the drug are provided for up to seven days. When she is handsome, she can avoid the necessary pain and infection. In theory, _ _ _ _ _ may be beneficial in the delivery of many therapeutic eggs, _ protein f gastrointestinal decomposition and poor gastrointestinal absorption. Such as interference with eggs from fine blood clearance, and in order to make it blood The degree is maintained at a high value, and needs to be delivered at a sustained rate. The U.S. patent case number and the transdermal device are easy to shoot on the (4). 5,814,599) 〇In spite of these advantages, the skin is sealed by a skin. It has low penetrability and is currently only very rare in clinical applications and there are no proteins or peptides to be administered in the form of percutaneous 4 201103574. This low permeability is due to the stratum corneum (sc) (which is The outermost layer of the skin, caused by the flat, dead cells (keratinocytes) filled with keratinous fibers of the lipid bilayer membrane. The height-regular arrangement of the lipid bilayers provides sc-impermeable properties (US Patent No. 5,814,599). Good method. [Summary of the Invention] Transdermal drug delivery provides an advantageous method of administration instead of sputum delivery and injection. However, the skin substance can be worn by the phase #low, so its application is limited to Drugs. Therefore, there is still a need to propose a solution to the aforementioned shortcomings and inadequacies in the art, especially in combination with a method for improving transdermal drugs.
，該生物活性劑包括 於本發明之又1具體態樣， 以上類型之生物活性劑。於本發明之 活性劑含註少—種親水性生物活性The bioactive agent comprises a further embodiment of the invention, a bioactive agent of the above type. The active agent of the present invention contains a small amount of hydrophilic biological activity
劑。於本發明之 •’該生物 又另一項 201103574 瓜^/明之另—項具體態樣，該蛋白分解酵素係選自由木 :蛋白％、_抑制劑、無花果蛋白酶、難蛋白酶、彈力蛋 =蛋白酶、透明質酸酶、鏈激酶、鏈球菌去氧核糖核酸 轰夷蛋白酶、胰纖_、_凝_酶、㈣粉酶、 糖核酸酶、膠原蛋白酶、枯草_及其任意組合所組成 ^本發明之另一項具體態樣，該蛋白分解酵素係遭度小於 ”J、於50、40或30) μδ/ηι1之木瓜蛋白酶。 方面’本發明係關於一種改進並增強生物活性劑遞 給藥之遞送方法。該方法包含將有下列所示物之有效 ⑷脂質體⑽封裝㈣脂質體中之生物活性劑； (:)夕數經共輛於該脂質體表面上之木瓜蛋白酶分子；及⑼技 «上可接受之載體的組成物給予皮膚， 通過皮膚之功效者。敲衫糊達成遞送 糾-方面’本剌侧於—_於生物活性劑遞送 ;之性劑，（c)多數經共輛於該脂質體表面上之蛋白 二解酵素分子；及⑻醫藥上可接受之载體，其中該蛋白 係選自由木瓜蛋白酶、胰酶抑·、無花果蛋_、渡蘿蛋 P、彈力蛋白酶、胃蛋白酶、透明f_、鏈激酶、 去氧核糖核酸酶、胰蛋白酶、騰凝乳蛋白酶、α韻乳蛋白酶、 6 201103574 菌酶及其任意 a -¾泰酶、去氧核糖核酸酶、膠原蛋白酶、枯草 組合所組成之組群。 鄕體·，频祕可進 於該脂質體中之太半斜私廿山 乂匕3經包圭 脂質體t之物__懷於位糊 、不未微粒内。該奈米微粒可為聚(乳酸-共 (PLGA)或水凝勝奈米微粒。 乙転^ 本發明之此等其他方面由下列較佳具體態樣，結合下列圖 彰顯出，_在不偏離該揭示内容之新穎概念 神及犯圍下’可於射進行變更與修改。 …所提供之圖式例舉·本發明之—或多項具體態樣，其與 下述坪細說明-起’係用轉釋本發明之創作原理。若可能， 於圖式中細叫考編號，來引述實施例中之烟或類 件。 、 【實施方式】 在本發明所使用的特殊術語有其原本的意義，如下所用的 某些特殊術語是提供熟悉該技藝者能更進—步了解本發明内 合。為了方便起見-些特殊術語將會使用斜體字或引號標示出 來，但這些被標示出來的部分並不會影響到特殊術財身的範 圍或意義，就如同在本文中未被標示的文字一樣，也就是說同 樣的事情會有-個以上的說法。本剌之其他特色及優點將於 下列實施範射被進-步舉例與綱，而該實祕例僅作為輔 201103574 助說明，並非用於限制本發明之範圍。 除非另有規定，本發明所涉及的科學和技術所用詞囊和一 般普通技能所使用的詞彙為相同的，若是有所衝突的情况下， 本發明將會給予名詞新的定義。 本發明所使關“約略”、“大約，，或“大概，，廣義而言 應意指’所給予數值或範圍之上下百分之二十，較佳是百分之 十’且更佳地是百分之五的範圍内，在此所給予之數值數量為 大約的意思，表示術語“約略，，、“大約，，或“大概，，並沒有 明文強制規定。 實施例 無意於限制本發明之範圍，以下將呈現根據本發明各種實 施例之例舉性儀ϋ、裝置、方法和細關結果。應注意，實施 例中為了方便讀者閱讀所使用的標題或副標題，並不以任何方 式限制本發明的範圍。此外，在本文中所提出和披露的某些理 論’但無論他們是對奴錯，只彻咐根縣發明所實施 的，而不需考慮任何特定的理論或行動的計晝，都應被限制在 本發明的範圍之内。 實施例1:封裝有生物活性劑之载劑的製備 封裝有羧基螢光素之脂質體 將U-二油酿基,甘油㈣醯乙醇㈣備義基），或 /醯基ΡΕ (一種市售可得含有幾基基團之碌脂質）用於與 瓜蛋白酶進行偶合。將琥賴基ΡΕ、以二硬脂酿基*甘 $ 201103574 油-3-雜膽驗(DSPC)與膽固醇，以1:9:3之重量比例一起混合 於置於大約溫水浴之氣仿溶財。接著藉由去除溶劑而形 成薄的脂質薄膜。 將絲螢光素加至該薄的脂質薄膜。將所成之混合物於大 約6〇 C之水洛中擾拌約一小時。然後將該混合物推擠通過小型 擠出機（渡片孔徑：0.2 ，20次），接著通過SephadexG_25 管柱將封裝有CF的脂質體分離。 封裝有奈米微粒之脂質體 利用與前述類似之程序製備得封裝有聚(乳酸_共_乙醇酸) (PLGA)奈米微粒的脂質體，惟其係使用pLGA奈米微粒取代 CF與該脂質薄膜水合。 封裝有透明質酸之脂質體 利用與前述類似之程序製備得封裝有透明質酸的脂質 體准其係使用具有分子量大約8〇〇kDa的經螢光素標記之透 月貝酉夂取代CF。藉由在封裝作用後將該脂質體溶解，並測量 攸月曰貝體釋放出馳螢光素標記之透明質賴螢絲度，而測 疋得封裝於脂質體中之透明質酸量。 封裝有維生素£與輔酶q1〇之脂質體 利用與前述類似之程序製備得封裝有維生素£與輔酶Q1〇 的脂質體，惟其係將DSPC、琥珀醯基？£、膽固醇、維生素£ 與輔_Ql〇，以5:3:1:1:1之莫耳比例混合於氯仿溶液中。脂質 體中維生素E與輔酶Q1G的包含量，係藉由使用她处15〇 χ 201103574 4 mm RP-18管柱及UV _器’流速為大約Q 6讀响之高效 能液相層析術(HPLC)測定得。 封裝有透明質酸之脂質體 材料。1，2-二硬脂醯基，甘油_3屬酿膽驗卿^❻ 二油醯基-sn-甘油-3-_乙醇胺_N__酿基）（號鋪基pE) 係購自Avanti Polar Lipids (阿拉貝斯特，从）。葡萄糖分析 套組係購自BioVision (朗山，CA)。其他所有化學品皆講 自西格瑪化學公司（聖路躺，_>17_室係購自p⑽如 (荷樂市，PA)。 方法。以薄膜水合方法製備得脂質體。簡言之，將脂質化 合物DSPC、琥細基PE與膽固醇，以ι 〇:〇 ι:〇.7之莫耳比例 混合於氣仿中，並藉由旋轉蒸發作用將溶劑去除，而在圓底燒 瓶上形成脂質薄膜。再將脂質薄膜於真空下乾燥過夜，以去^ 紐，有機溶劑。將一毫升⑽_葡萄糖加入以水合2〇啤 脂f薄膜，並將動旨㈣浮_持於下1辦。待水合作 用凡成後’將物脂質雜浮錢行超音波震盪1G分鐘。使 料型擠出機U刪iPGlarLipids)藉由於⑽下推擠通過聚 =j片⑽、）20次而取得脂質體之粒徑。任何未被封 、、葡喃係經由’以sephadexG_25管柱⑽讀生物科學 AB)進行之凝膠過濾法而將其從脂質體移除。 實施例2.附加蛋白分解酵素之藥物载劑的製備 木瓜蛋白酶共軛於脂質體之表面上 201103574 藉由使用偶合劑鹽酸1-乙基-3-[3-二甲胺基丙基]羰二亞醯 胺（EDC ’西格瑪化學公司）（0.2 M)，而達成木瓜蛋白酶化合 物（1 mg)之一級胺基基團與脂質體（2χ 1〇]2脂質體/ml)之 羧基基團的共軛作用。偶合反應110係於磷酸鹽緩衝食鹽水 中’於室溫下進行2小時。附加木瓜蛋白酶之脂質體係經由， 使用尺寸排阻層析術管柱（Bi〇SepSEC_S2〇〇〇，非諾梅尼斯） 之HPLC (间效能液相層析術）進行純化，而去除未反應的木 瓜蛋白酶。 圖1為顯不木瓜蛋白酶分子1〇4與，位於脂質體的脂質雙 層表面108上之縣1〇6，難EDC偶合反應11〇進行偶合的 不意圖。EDC為-種羧基與胺_反應性零_長度交聯劑。咖首 先與絲106反應並形成胺反應性〇_醯基異脲_間物，其很快 地與胺基反應而形成醯胺鍵。EDC在此係用於將木瓜蛋白酶 104之胺基連結至位於脂質體表面上的羧基⑽。 木瓜蛋白酶1〇倾認為是，用於增加特定藥物分子1〇2通 過皮膚孔洞2〇4之通透性增進劑（圖。木瓜蛋白酶綱本 身太大而無法通過活的表皮彻。‘然而已f經提出關於其不安 定性及安全濃度等議題。圖2B例舉說明利用微米化或奈米化 藥物载劑搬（例如，脂質體）將生物活性分子1〇2濃缩於节 載劑内，及位於其表面上之木瓜蛋白酶104用來增進及延的 生物活性分子搬通過皮膚2⑽的孔洞2G6之運送。1麵在 於藉由將木瓜蛋白酶⑽與生物活性分子102濃縮，可辦強唾 201103574 皮遞送效率並減少溶液中所需要木瓜蛋 :能夠降低__木終响_編_副口 木瓜蛋白酶之定量 =_ _ _ (例如〒肖基苯醯 疋瓜蛋白酶之總量。將顧升溶於二甲亞硬__ 之受質溶液與]65 μ1之〇」％鱗酸鹽緩衝食鹽水卿， PH Μ混合。觀合胁抓下料5錄爾瓜蛋白酶或 =木瓜蛋白酶之脂質體溶邮⑼加人（木瓜蛋白酶濃度範 圍介於0.25 mg/m】至2吨⑽’並將其持續於坑下培育如 分鐘。藉由添加15 之3 N鹽酸來終止反應。於細下記 錄樣权吸光值。將樣本溶液之木瓜蛋自酶濃度與自由態木瓜 蛋白酶之標準曲線配合。為避免脂質體的干擾，將所有樣本溶 、、進行吸光值測;，先以αΐμπι濾膜（密里泊(遍p〇re》 過渡。猎由_之定量方法測量各含有木瓜蛋白酶-共輛脂質體 知識管中的總木瓜蛋白酶濃度。 經共輕於單一脂質體上之木瓜蛋白酶分子數量 错由將木瓜蛋白酶分子總數(N木瓜蛋白酶)除以脂質體之總數 (N_)’而計算得共輛至單一脂質體上之木瓜蛋白酶分子數量 (I): N木瓜蛋白酶/N脂質理 術語N木瓜蛋㈣表示以顯色受質測得之木瓜蛋白酶分子總 數。術語N _表示脂質體之總數，其係藉由將碟脂質分子總 12 201103574 數除以存在單一、200 nm脂質體中之填脂質分子數（大約16 xlO5磷脂質分子/脂質體）而計算得。磷脂質之濃度係藉由如下 所述之比色法進行測量：將27.03克六水合氣化鐵(FeCl36H2〇) 與30.4克硫氰酸録(nhjcn)溶於丨L去離子水中，而製得鐵 硫氰酸銨之儲備溶液。製成磷脂質濃度之標準校正曲線。將各 別磷脂質樣本與等體積之鹽溶液劇烈混合一分鐘。將有機相與 水相分離，並以UV/Vis記錄吸光值’以配合得樣本的磷脂質 濃度。 、 計算每單一經共軛脂質體之有效木瓜蛋白酶濃度 對於每一經酵素共軛的脂質體，其相對應有效酵素濃度之 计算如下：計算單—脂質體的總表面積（A = 4AR2)。假設木 瓜蛋白酶是均自分布於脂·表面上，兩木瓜蛋_分子間之 距離等於d =，/ A,/2。其中”η”代表每-脂質體之木瓜蛋白酶 數量’而”Α”係指月旨質體的表面積。一個木瓜蛋白酶分子所佔 據之體積為d。計算得有效木瓜蛋白酶濃度料木瓜蛋白酶總 分子數每升。域3所示，每―崎體之有效木瓜蛋白酶漢度 係與所接附的木瓜蛋白酶分子數量成正比。 以位於350木瓜蛋白酶分子/脂質體之X轴數值為例，相 =應之有效木瓜蛋白酶濃度為245咖。而於视木瓜蛋白酶 知胃體下3後木瓜蛋自酶共碰㈣之試管中的始 =蛋白酶濃度為。因此，每—脂倾之有效木終 白酶濃度實際上增加倍㈤/U8 =观），此係由於事實 13 201103574 上木瓜蛋白酶分子被濃縮於脂質體的表面上所致。 實施例3:經封裝之生物活性劑的經皮遞送 使用活體外Franz擴散室評估附加蛋白分解酵素之藥物載 劑的經皮穿透功效。簡述之，將一片使用電動植皮刀（zimmer Inc·，USA)切成厚度達500 μιη之豬腹部皮膚組織，架設於垂 直式Franz擴散室的兩個半室之間（R=丨5 mm，皮膚面積：丨.77 cm2 ’接受器體積：12ml)並以夾子固定。 於各種不同經皮藥物定送模式測量通透效率。測試下列四 種不同的藥物定送模式：（1)表面上附加木瓜蛋白酶之封裝有 葡词糖的月曰貝體（350木瓜蛋白酶分子/200 脂質體，纯木瓜 蛋白酶26 ，總葡萄糖5 ^ ; (2)自由態木瓜蛋白酶(5〇〇Agent. In the present invention, 'this organism is another 201103574 melon ^ / Ming other - item specific, the proteolytic enzyme is selected from wood: protein%, _ inhibitor, ficin, difficult protease, elastic egg = protease , hyaluronidase, streptokinase, streptococcus deoxyribonucleic acid protease, pancreatic fiber _, _ clotting enzyme, (four) powder enzyme, nuclease, collagenase, hay _ and any combination thereof In another embodiment, the proteolytic enzyme is less than "J, 50, 40 or 30" μδ/ηι1 of papain. Aspects of the invention relate to an improvement and enhancement of bioactive agent delivery a method of delivery comprising the following: (4) liposome (10) encapsulating (iv) a bioactive agent in a liposome; (:) a papain molecule co-hosted on the surface of the liposome; and (9) «The composition of the acceptable carrier is given to the skin, through the effect of the skin. Knocking paste to achieve delivery correction - aspect 'Ben 剌 side --- for bioactive agent delivery; sex agent, (c) majority of vehicles Liposomal surface a protein lysin molecule; and (8) a pharmaceutically acceptable carrier, wherein the protein is selected from the group consisting of papain, pancreatin, fig egg, ferry egg P, elastase, pepsin, transparent f_, a group consisting of streptokinase, deoxyribonuclease, trypsin, chymotrypsin, alpha-prolactin, 6 201103574 zymase and any combination of a-3⁄4 tyrosin, deoxyribonuclease, collagenase, and hay鄕 · , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Poly(lactic acid-co-(PLGA) or hydrogel-compound nano-particles. 乙転^ These other aspects of the invention are illustrated by the following preferred embodiments in combination with the following figures, _ without departing from the novel concept of the disclosure The sacred and sacred stipulations may be changed and modified by the sequel. The exemplification of the drawings provided by the present invention, or the specific features of the present invention, and the following detailed descriptions The principle of creation. If possible, the number is called in the drawing. Reference is made to the smoke or the like in the embodiments. [Embodiment] The specific terms used in the present invention have their original meanings, and some specific terms used as follows are provided to those skilled in the art to further understand the present invention. For convenience - some special terms will be marked in italics or quotation marks, but these marked parts will not affect the scope or meaning of the special wealth, as in this article. The same text is marked, that is to say, the same thing will have more than one statement. The other features and advantages of Benedict will be taken into the following examples and examples, and the actual case is only used as a supplement to 201103574. The description is not intended to limit the scope of the invention. Unless otherwise specified, the words used in the science and technology of the present invention are the same as those used in the general skill, and if there is a conflict, the present invention will A new definition of noun will be given. The present invention is used to mean "about", "about," or "about, broadly, means that the value or range given is less than twenty percent, preferably ten percent, and more preferably. In the range of five percent, the numerical value given herein is approximately the meaning of the term "about,", "about," or "presumably, and is not expressly mandatory. The examples are not intended to limit the invention. Ranges, exemplifications, devices, methods, and succinct results in accordance with various embodiments of the present invention will be presented below. It should be noted that the headings or subtitles used in the examples are not limited in any way for the convenience of the reader. The scope of the invention. In addition, some of the theories presented and disclosed herein, but whether they are slaves, are only implemented in the roots of the invention, without regard to any particular theory or action. All should be limited to the scope of the present invention. Example 1: Preparation of carrier encapsulating bioactive agent Preparation of liposome encapsulated with carboxyfluorescein U-dioleic glycerol, glycerol (tetra) hydrazine (IV) Yiji ), or / 醯 ΡΕ (a commercially available lipid containing several groups of groups) for coupling with chymotrypsin. Acryl hydrazine, with distearin broth * Gan $ 201103574 oil -3- The cholesterin test (DSPC) and cholesterol are mixed together in a weight ratio of 1:9:3 in a gas bath placed in an approximately warm water bath, and then a thin lipid film is formed by removing the solvent. To the thin lipid film, the resulting mixture was turbulent for about one hour in about 6 ° C. The mixture was then pushed through a small extruder (aperture aperture: 0.2, 20 times), followed by Sephadex G_25 column separates liposomes encapsulated with CF. Liposomes encapsulated with nanoparticulates Liposomes encapsulated with poly(lactic-co-glycolic acid) (PLGA) nanoparticles are prepared using procedures similar to those described above, except The pLGA nanoparticle is substituted for CF to hydrate with the lipid film. The liposome encapsulated with hyaluronic acid is prepared by a procedure similar to the foregoing to obtain a liposome encapsulated with hyaluronic acid using a molecular weight of about 8 〇〇 kDa. Fluorescein-labeled夂 Substituting CF. The hyaluronic acid encapsulated in the liposome is measured by dissolving the liposome after encapsulation and measuring the release of the fluorescein-labeled hyaluronic fluorescein. The amount of acid. Liposomes encapsulated with vitamin £ and coenzyme q1〇 were prepared using a similar procedure as described above to prepare liposomes encapsulated with vitamin £ and coenzyme Q1〇, except for DSPC, amber, cholesterol, vitamins and Auxiliary _Ql〇, mixed in a chloroform solution at a molar ratio of 5:3:1:1:1. The amount of vitamin E and coenzyme Q1G in the liposome is 15 〇χ 201103574 4 mm RP by using her The -18 column and UV _ 'flow rate was measured by high performance liquid chromatography (HPLC) of approximately Q 6 read. A liposome material encapsulated with hyaluronic acid. 1,2-distearyl, glycerol _3 genus cholera qing qing ❻ ❻ 醯 醯 - - s 甘油 甘油 甘油 甘油 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) A A A A A A A A A A Lipids (Arabister, from). The glucose analysis kit was purchased from BioVision (Langshan, CA). All other chemicals are from Sigma Chemical (St. Lu's, _> 17_ room purchased from p (10) such as (Holly, PA). Method. Preparation of liposomes by membrane hydration. Briefly, lipids Compound DSPC, acryl PE and cholesterol were mixed in a gas mixture at a molar ratio of ι 〇: 〇ι: 〇.7, and the solvent was removed by rotary evaporation to form a lipid film on a round bottom flask. The lipid film is then dried under vacuum overnight to remove the organic solvent. One milliliter of (10)-glucose is added to hydrate the 2 〇 〇 f f film, and the purpose (4) floats to hold the next one. After using the singularity, the squirting of the lipids and the floating liquids for 1G minutes. The granules of the liposomes were obtained by pushing the slabs (10), 20 times by pushing (10). path. Any unblocked, glucosamine was removed from the liposomes by gel filtration by 'sephadex G_25 column (10) reading Biosciences AB). Example 2. Preparation of a drug carrier with additional proteolytic enzymes Papain conjugated to the surface of a liposome 201103574 by using a coupling agent 1-ethyl-3-[3-dimethylaminopropyl]carbonyl Amidoxime (EDC 'Sigma) (0.2 M), and a total of one amino group of papain compound (1 mg) and a carboxyl group of liposome (2χ122 liposome/ml) Yoke effect. The coupling reaction 110 was carried out in phosphate buffered saline for 2 hours at room temperature. The papain-added lipid system is purified by HPLC (inter-performance liquid chromatography) using a size exclusion chromatography column (Bi〇SepSEC_S2〇〇〇, Fenomenis) to remove unreacted papaya Protease. Fig. 1 is a schematic diagram showing that the papain molecule 1〇4 and the county 1〇6 located on the lipid double layer surface 108 of the liposome are coupled with a difficult EDC coupling reaction. EDC is a carboxyl group and an amine-reactive zero-length crosslinker. The coffee head first reacts with the silk 106 and forms an amine-reactive 〇-mercaptoisourea-interstrate which rapidly reacts with the amine group to form a guanamine bond. EDC is here used to link the amino group of papain 104 to a carboxyl group (10) located on the surface of the liposome. Papain 1 is considered to be a permeability enhancer for increasing the specific drug molecule 1〇2 through the pores of the skin 2 (Fig. The papain class itself is too large to pass the live epidermis. 'However Issues such as its instability and safe concentration have been proposed. Figure 2B illustrates the use of micronized or nanochemical drug carriers (eg, liposomes) to concentrate the bioactive molecule 1〇2 in the carrier, and The papain 104 on its surface is used to enhance and extend the transport of bioactive molecules through the pores 2G6 of the skin 2(10). One side is to concentrate the papain (10) with the bioactive molecule 102 to deliver the strong saliva 201103574. Efficiency and reduce the need for papaya eggs in solution: can reduce the __ wood end ring _ _ _ _ _ _ _ _ _ _ (for example, the total amount of 〒 基 醯疋 醯疋 醯疋 醯疋 。 。 。 。 将 顾 顾Hard __ the solution of the solution and the [65 μ1 of the 〇"% sulphate buffer saline, PH Μ mixed. Guanhe threatened to take the material 5 guagua protein or = papain liposome leaching (9) plus people (Papain Concentrate The range is from 0.25 mg/m to 2 tons (10)' and it is continued under the pit for centrifugation. The reaction is terminated by adding 15 of 3 N hydrochloric acid. The sample absorbance is recorded under the sample. The egg self-enzyme concentration is matched with the standard curve of free-form papain. In order to avoid the interference of liposome, all the samples are dissolved and the absorbance is measured; first, the αΐμπι filter (Milipo (trans-p〇re) transition). Hunting is a quantitative method for measuring the total papain concentration in each of the papain-containing liposome knowledge tubes. The total number of papain molecules on a single liposome is determined by the total number of papain molecules (N papain). Divided by the total number of liposomes (N_)' to calculate the number of papain molecules on a single liposome (I): N papain / N lipid theory term N papaya egg (four) means measured by color rendering The total number of papain molecules. The term N _ indicates the total number of liposomes by dividing the total number of disc lipid molecules 12 201103574 by the number of lipid-filled molecules present in a single, 200 nm liposome (approximately 16 x lO5 phospholipids) Calculated by molecular/liposome. The concentration of phospholipids was measured by the colorimetric method as follows: 27.03 g of iron hexahydrate hexahydrate (FeCl36H2〇) and 30.4 g of thiocyanate (nhjcn) were dissolved.丨L deionized water to prepare a stock solution of ammonium iron thiocyanate. Prepare a standard calibration curve for phospholipid concentration. Mix the individual phospholipid samples with an equal volume of salt solution for one minute. Phase separation, and record the absorbance value in UV/Vis to match the phospholipid concentration of the sample. Calculate the effective papain concentration per single conjugated liposome for each enzyme-conjugated liposome, the corresponding effective enzyme The concentration was calculated as follows: Calculate the total surface area of the mono-liposome (A = 4AR2). Assuming that papain is self-distributed on the lipid surface, the distance between the two papaya eggs is equal to d =, / A, /2. Wherein "η" represents the number of papain per liposome and "Α" refers to the surface area of the plastid. A papain molecule occupies a volume of d. Calculate the effective papain concentration of the total number of molecules of papain per liter. As shown in the domain 3, the effective papain system of each of the Saskatchewan is directly proportional to the number of molecules of the papain attached. Taking the X-axis value of the 350 papain molecule/liposome as an example, the phase = effective papain concentration is 245 coffee. However, the concentration of protease in the test tube of the papa egg from the enzyme (4) was determined by papain. Therefore, the concentration of the effective wood terminase per phlegm is actually increased by a factor of (5) / U8 = guan), which is due to the fact that the papain molecule is concentrated on the surface of the liposome. Example 3: Transdermal delivery of encapsulated bioactive agents Transdermal penetration efficacy of drug carriers with additional proteolytic enzymes was assessed using an in vitro Franz diffusion chamber. Briefly, a piece of pig skin tissue with a thickness of up to 500 μm was cut into a 500 μm thick pig skin tissue using a motorized skinning knife (zimmer Inc., USA), placed between two halves of a vertical Franz diffusion chamber (R=丨5 mm, Skin area: 丨.77 cm2 'receiver volume: 12 ml) and fixed with a clip. Permeability was measured in a variety of different transdermal drug delivery modes. The following four different drug delivery modes were tested: (1) the addition of papain to the surface of the scorpion scorpion (350 papain molecule / 200 liposome, pure papain 26 , total glucose 5 ^; (2) Free state papain (5〇〇
Kg/ml) +自由態葡萄糖(5慮）；⑶自由態葡萄糖(5碰广及 自由態木瓜蛋白酶(5〇〇 pg/mi) +封裝有葡萄糖的脂質體卩 mM)。 將得自各組之樣本（每樣本取1 ml)加至供者隔室中。將 接受隔室充滿0.1 MPBS。每30分鐘進行採樣達3小時，並以 葡萄糖分析套組（BioVisi〇n c〇)根據製造商之指示（tecan， InifimtelVQOO)進行分析。所有實驗皆於室溫下完成。 所計算得每一經附加脂質體之木瓜蛋白酶總量為〇. 〇 2 5 m_，相當於350木瓜蛋白酶分子/脂質體，或相當於有效木 瓜蛋白酶濃度為5.6 mg/m卜經報導木瓜蛋㈣之安全濃度為 約 〇.039 mg/ml 左右，於溶液中等於 4 μΜ (US005534260A)。 14 201103574 表1*Kg/ml) + free glucose (5); (3) free glucose (5 hits and free papain (5〇〇 pg/mi) + liposome encapsulated with glucose mM mM). Samples from each group (1 ml per sample) were added to the donor compartment. Fill the receiving compartment with 0.1 MPBS. Samples were taken every 30 minutes for 3 hours and analyzed by the glucose analysis kit (BioVisi〇n c〇) according to the manufacturer's instructions (tecan, Inifimtel VQOO). All experiments were done at room temperature. The total amount of papain calculated for each additional liposome is 〇.25 m_, which is equivalent to 350 papain molecules/liposomes, or equivalent to an effective papain concentration of 5.6 mg/m, which is reported to be papaya eggs (4). The safe concentration is about 039.039 mg/ml, which is equal to 4 μΜ (US005534260A) in solution. 14 201103574 Table 1*
* ··各樣本中之總葡萄糖為5mM。 木瓜蛋白酶月曰質體@葡萄糖溶液中之總木瓜蛋白酶濃产 為25eg/ml。符號，，@，，代表“封裝”。 又 ***:自由態木瓜蛋白酶之濃度為5〇〇μδ/ιηΐ。 比較不同遞送模式間的葡萄糖穿皮效率。於下列不同條件 下獲得葡萄糖通透性(mg/hr):⑴葡萄糖經封裝於木瓜蛋白酶- 共輛脂質體心（2)葡萄糖藉由自由態木瓜蛋白酶單獨⑼倍 濃度鋪萄糖混合）進行遞送（表1);及⑶制糖經封裝於 脂質體内並藉由自由態木瓜蛋白酶進行遞送（表以通透性⑺ 係由P = ;/Cv計算得，其中，，：，，代表每單位面積每小時流通過 皮膚屏障之葡萄糖量(mg/cm2xhr)，而，,Cv,，代表生物活性分子 之濃度(mg/cm3)。 201103574 結果表示，當生物活性分子經包褒入木瓜蛋白酶—共輕脂 質體内時，該分子之經皮遞送效率顯著較該等藉由自由態木瓜 蛋白酶(500 pg/ml)單獨進行遞送者增加。儘管事實上於:加木 瓜蛋白酶之脂質體溶液中的總木瓜蛋白酶濃度只有自由態可 溶木瓜蛋⑽的二十分之-（亦即25 ，仍可__ 由木瓜蛋白酶-共姉諸咖所猶之經皮藥物遞送。結果 顯示，與脂質體飾之木瓜蛋白酶_皮遞送效雜未經共輕 者高20倍。 表2** ·· The total glucose in each sample is 5 mM. The total papain production in papain ruthenium @glucose solution was 25eg/ml. The symbol, @,, stands for "package". ***: The concentration of free papain is 5〇〇μδ/ιηΐ. Glucose transdermal efficiency was compared between different delivery modes. Glucose permeability (mg/hr) was obtained under the following conditions: (1) glucose was encapsulated in papain - a total of liposome hearts (2) glucose was delivered by free papain alone (9) concentration of glucose mixed) (Table 1); and (3) The sugar is encapsulated in the liposome and delivered by free-state papain (the table is calculated by permeability (7) from P = ;/Cv, where:,:, represents per unit The amount of glucose per hour through the skin barrier (mg/cm2xhr), and, Cv, represents the concentration of the bioactive molecule (mg/cm3). 201103574 The results indicate that when the bioactive molecule is encapsulated into papain - a total In light liposomes, the transdermal delivery efficiency of this molecule is significantly increased compared to those delivered by free papain (500 pg/ml), despite the fact that: total in the liposome solution with papain The papain concentration is only 20% of the free-form soluble papaya egg (10) - (ie 25, still __ is delivered by papain - co-medicine transdermal drug. The results show that it is decorated with liposomes papain_ Heteroaryl delivery efficiency without the light 20 times were high. TABLE 2 *
木瓜蛋白酶-脂質體@葡萄糖溶液中之總木瓜蛋白酶濃度 為25μδ/ηύ。符號，，@”代表“封裝”。 /又 :各樣本中之總葡萄糖為5 mM。 .自由態木瓜蛋白酶之濃度為500 pg/ml。 16 201103574 别迷對於本發明較佳實施例之描述，僅為用以說明及敛述 的目的’而非用以限制本創作。熟悉該技藝者將可體會本創作 可能使用於很多形式、結構和材料的修改。 所選擇及贿之具縣樣與實制，係為轉本發明之原 理及其實_制，贿熟驗微藝者能夠顧本發明及各 種實施態樣，且驗其所職的特期途進行各種適合之修 改。其他具體實施例在不脫離本發明之精神及範圍下，對於熟 悉該項技藝者是顯4知的。因此，本發明之細應由後附申、 睛專利細所界定’而非受限於前述的·及實施例。 於本發縣财個及論述某些參考讀，其可包括專利 案、專獅請案及各種公開文獻。此等參考文獻之引用及論述 僅用以闡明本發明之說明，而非認可此類文獻係屬本發明内容 之“先前技藝，，。所有於本說明書中引用及論述之參考文獻， 係完整地以引用方式納入本文’且擴張至其各別引用之參考文 獻。 【圖式簡單說明】 圖1為顯示封裝有生物活性分子之脂質體(A)與木瓜蛋白 酶（由箭號指示）進行EDC偶合反應，形成木瓜蛋白酶-共輕 之脂質體(B)的示意圖。 圖2A為生物活性分子1〇2經由蛋白分解酵素，例如呈自 由態之木瓜蛋白酶104做為穿透增進劑進行經皮遞送示意圖。 圖2B為生物活性分子1〇2被包裝於其表面上接附有如圖 201103574 2A之蛋白分解酵素谢的脂 圖3顯示各脂質體之有致木二進行經皮遞送示意圖。 木瓜蛋白酶分子數目成正比。 祕度與接附於其上之 2顯示軸糖於有或無自域木瓜蛋 輕之木錢自畴訂稍歧讀纽=貝體共 【主要元件符號說明】 的時W化。 102生物活性分子 104木瓜蛋白酶 106羧基 108脂質雙層表面 110偶合反應 202藥物載劑 204皮膚孔洞 206孔洞 208皮膚 18The total papain concentration in the papain-liposome@glucose solution was 25 μδ/ηύ. The symbol, @" stands for "package". / Again: the total glucose in each sample is 5 mM. The concentration of free state papain is 500 pg/ml. 16 201103574 Not to be described in the preferred embodiment of the invention, It is for illustrative purposes only and is not intended to limit the creation. Those skilled in the art will appreciate that the creation may be used in many forms, structures, and materials. The system is to transfer the principle of the invention and the actual system, and the person who is interested in the micro-art can make various modifications according to the invention and various implementation aspects, and examine the special period of his position. Other specific examples are Without departing from the spirit and scope of the present invention, it will be apparent to those skilled in the art. Therefore, the details of the present invention should be defined by the appended claims and the details of the patents, and are not limited by the foregoing. In the case of Benfa County, and in the discussion of certain reference readings, which may include patents, lions, and various public documents, the citation and discussion of such references are only used to clarify the description of the present invention, rather than Such literature belongs to the invention The content of the "previous skills,,. All references cited and discussed in this specification are hereby incorporated by reference in their entirety in their entirety in their entirety in their entirety herein BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing an EDC coupling reaction of a liposome (A) encapsulating a bioactive molecule and a papain enzyme (indicated by an arrow) to form a papain-co-lipid liposome (B). Figure 2A is a schematic representation of transdermal delivery of a bioactive molecule 1〇2 via a proteolytic enzyme, such as papain 104 in a free state as a penetration enhancer. Fig. 2B is a diagram showing that the bioactive molecule 1〇2 is packaged on its surface and attached with a proteolytic enzyme as shown in Fig. 201103574 2A. Fig. 3 shows a schematic diagram of transdermal delivery of each of the liposomes. The number of papain molecules is proportional. The secretness and the attached to the 2 display shaft sugar in the presence or absence of self-domain papaya egg light wood money from the domain to read a little read the new = shell common [main symbol description] time W. 102 bioactive molecules 104 papain 106 carboxyl 108 lipid bilayer surface 110 coupling reaction 202 drug carrier 204 skin pores 206 holes 208 skin 18