200409656 玖、發明說明 【發明所屬之技術領域】 關 不知月疋有閉々;一裡笊不顆粒及具製造方法，且特別 於一種可應用於包埋化學物質的奈米顆粒及其製造方法 【先前技術】 利用水溶性物質或是水不溶性物質製造奈米顆粒，從最早 的相分離（C〇acervation)*法到最近幾年所使用的均質 (homogenization)方法。奈米顆粒在食品業、製藥業、、化妝 品及半導體晶圓製程材料上的應用已被廣泛地研究。奈米顆粒 必須是純化的極微小顆粒，上述兩種傳統方法必須使用大量的 有機溶劑和界面活性劑，不但有殘餘的問題，而且方法本身製 程繁瑣、控制條件複雜及機器設備昂貴不易取得。 至今奈米顆粒的合成方法有很多，每一種方法都各有其優 點、缺點及限制，現今的技術所面臨的共同挑戰是如何能改進 製程以降低生產成本及符合商業化量產要求。另外如何有效使 製造奈米顆粒過程中，不使用掺雜物的情況下，又可有效地合 成具有穩定相的奈米顆粒，是現今十分重要的 例™為使用均質舆相分離兩研種=它利 用過度冷卻溶化物質（如磷脂質）為包埋物，被包埋物可能是藥 劑、殺蟲劑或是肥料等，#入安定劑可為介面活性劑和乳化劑 一起混合，運用循環均質如壓力1200bar循環10次或是離心" 攪拌如轉速4000rpm、20分鐘，可產生30 nm〜5〇〇⑽的顆粒， 最後室溫冷卻。 例如US 6, 395, 302為使用相分離方法，它利用第一相聚 合物水相（如葡萄聚醣）、第二相乳化相（如微脂粒）與交又結合 聚合物（如甲基丙稀酸S曰）為包埋物，被包埋物是活性物質等， 200409656 在w/w加入微相有機溶劑一起混合攪拌，可產生1〇〇 〜ι〇〇 # m的顆粒，最後水洗與凍乾。 例如US 6, 395, 300為使用相分離方法，它利用多孔性物 質如親水的聚乙烯乙二醇或親油的磷脂質為包埋物，被包埋物 是低水溶性的藥劑等，加入固形劑如氨水與揮發溶液如丙酮、 氣仿一起混合擾拌，乳化為16,000rpm、5分鐘，可產生i〇〇nm〜5 的顆粒，然後使用噴霧乾燥除去揮發溶液（如6〇kg/hr、％ °C )，成品可作為藥錠與膠囊的原料。 例如US 6, 190, 692為使用相分離方法，它利用膨脹聚合 物如甲基纖維素或磷脂質為包埋物，被包埋物是藥劑或活性物 質等，加入鍵結作用的水溶液如聚乙烯化合物一起混合攪拌， 運用o/w方法產生有膜的顆粒，使藥劑產生控制釋放效果。 例如US 6, 387, 409為使用均質或微粒浮動床方法，它利 用如磷脂質為包埋物，被包埋物是水溶性或水不溶性的藥劑 等，加入安定劑如乙烯 '丙烯一起混合均質，可產生5〇 nm〜i〇 # m的顆粒。 例如US 6, 146, 663為使用均質乳化方法，它利用如磷脂 夤或乙烯聚合物為包埋物，加入分散劑如蔗糖一起混合均質乳 化，可產生100 nm的顆粒，然後使用孔洞220 nm的過濾膜過 濾去除細菌，最後凍乾成成品。 上述兩類油脂生產顆粒技術，其製造流程相當複雜，需要 控制的條件也相當多，如溫度變化、設備和配方，而且產率低、 非常耗時間及投資成本過高。再者，這兩種技術常需使用有機 溶劑，且需長時間在高溫下操作，因此不適合包覆菌種、疫苗 和蛋白質等物質。 除此之外，另有一種生產顆粒的技術被用來食品相關的顆 200409656 粒此技術主要疋將基質與被包埋物混合，再滴入液體中因而 產生顆粒。此技術通常不使用脂肪酸為載體，且產量很低，無 法大量工業化生產。 此外，台灣地窄人稠，傳統農業因需耗費土地及水資源， 所以現今慢慢沒落。且台灣於·2年初加人㈣後，國内水 產品的價袼大受打擊，使國内養殖業的養殖業者生存不易，所 =相關政府機構計劃能使台灣能夠成立亞太種苗繁殖中心，而 台灣的主要優勢在於繁養殖技術精良，可繁養殖之種類高達 種以上’且主要是高價海水養瘦魚種。石斑魚_直是屬於高價 魚種，且台灣近年已能自行人工繁殖，不過由於寸苗時常遭受 病毒性疾病的侵襲所以價格—直高居不下。虹彩病毒主要感染 ^斑f寸苗時期，且造成議以上的高死亡率，所以魚苗價格 一直高居不下。預估每次所買回的石斑㈣解化到三寸苗賣 出’其存活率約為30%。所以魚苗期的病毒性疾病感染的防治 一直為重要課題。 因此，若能以奈米顆粒以作為疫苗的傳送系統，特別是應 用於台灣石斑魚虹彩病毒感染症（Grouper Iridovirus 〇f200409656 发明, description of the invention [Technical field to which the invention belongs] There is a closed loop on the moon; there is no granule and a manufacturing method, and in particular, a nano granule applicable to embedding chemical substances and a manufacturing method thereof [ Prior art] Nanoparticles are manufactured using water-soluble or water-insoluble substances, from the earliest phase separation (Coacervation) method to homogenization methods used in recent years. The application of nano particles in the food industry, pharmaceutical industry, cosmetics and semiconductor wafer process materials has been extensively studied. Nanoparticles must be purified and extremely fine particles. The above two traditional methods must use a large amount of organic solvents and surfactants. Not only are there residual problems, but the method itself is cumbersome, the control conditions are complex, and the equipment is expensive to obtain. So far, there are many methods for synthesizing nano particles. Each method has its own advantages, disadvantages, and limitations. The common challenge facing today's technology is how to improve the process to reduce production costs and meet commercial mass production requirements. In addition, how to effectively make nano particles with stable phase without the use of dopants in the process of manufacturing nano particles is a very important example today. To separate two seeds using homogeneous phase = It uses excessive cooling to dissolve substances (such as phospholipids) as embeddings. The embeddings may be medicaments, pesticides, or fertilizers, etc. # 入定 剂 can be mixed with the surfactant and emulsifier, using circulating homogenization If the pressure is 1200 bar for 10 cycles or centrifugation, for example, the rotation speed is 4000 rpm for 20 minutes, particles of 30 nm ~ 500 ° F can be generated, and finally cooled at room temperature. For example, US 6, 395, 302 uses a phase separation method, which uses a first-phase polymer aqueous phase (such as glucosan), a second-phase emulsified phase (such as microlipids), and a cross-linked polymer (such as methyl). Acrylic acid (S) is an embedded substance, and the embedded substance is an active substance, etc. 200409656 Adding a microphase organic solvent together at w / w and mixing and stirring can produce particles of 100 ~ 100m, and finally washed with water With lyophilization. For example, US 6, 395, 300 uses a phase separation method, which uses porous materials such as hydrophilic polyethylene glycol or lipophilic phospholipids as embeddings, and the embeddings are low water-soluble agents, etc. A solid agent such as ammonia is mixed with a volatile solution such as acetone and aerosol, emulsified at 16,000 rpm for 5 minutes, and particles of 100nm ~ 5 can be produced, and then the volatile solution (such as 60kg / hr) is removed by spray drying. ,% ° C), the finished product can be used as raw materials for tablets and capsules. For example, US 6, 190, 692 uses a phase separation method, which uses a swelling polymer such as methylcellulose or a phospholipid as an embedding, the embedding being a medicament or an active substance, etc., and a bonding aqueous solution such as a polymer The ethylene compounds are mixed and stirred together, and the o / w method is used to produce particles with a film, so that the drug has a controlled release effect. For example, US 6, 387, 409 is a homogeneous or particulate floating bed method. It uses, for example, phospholipids as embeddings, and the embeddings are water-soluble or water-insoluble agents. Stabilizers such as ethylene and propylene are added to mix and homogenize. , Can produce 50nm ~ i〇 # m particles. For example, US 6, 146, 663 uses a homogeneous emulsification method. It uses, for example, phospholipids or ethylene polymers as embedding materials, and adds a dispersant such as sucrose to mix and emulsify homogeneously. It can produce 100 nm particles, and then use pores of 220 nm. The filter is filtered to remove bacteria, and finally lyophilized into a finished product. The above two types of oil and fat production granule technology have quite complicated manufacturing processes and require a lot of conditions to be controlled, such as temperature changes, equipment and formulations, and they have low yields, are very time consuming, and have high investment costs. Furthermore, these two technologies often require the use of organic solvents and long-term operation at high temperatures, so they are not suitable for coating bacteria, vaccines, and proteins. In addition, another technology for producing granules is used for food-related granules. 200409656 granules. This technology mainly mixes the matrix with the embedding material and then drips into the liquid to produce granules. This technology usually does not use fatty acids as a carrier, and the yield is very low, which cannot be mass-produced industrially. In addition, Taiwan is narrow and densely populated. Traditional agriculture consumes land and water resources, so it is slowly declining. In addition, since Taiwan was added in early 2nd year, the price of domestic aquatic products has been hit hard, making it difficult for the domestic aquaculture farmers to survive. Therefore, relevant government agencies plan to enable Taiwan to establish an Asia-Pacific seed breeding center, and The main advantage of Taiwan is that the breeding technology is excellent, and the species that can be breeded are up to more than one species. Grouper _ is a high-priced fish species, and Taiwan has been able to artificially propagate by itself in recent years, but the price is still high because the seedlings are often attacked by viral diseases. The iris virus mainly infects the spotted seedlings and causes a high mortality rate, so the fry prices have been high. It is estimated that each time the buyback of the grouper is reduced to three-inch seedlings sold, the survival rate is about 30%. Therefore, the prevention and control of viral disease infection at the fry stage has always been an important issue. Therefore, if nano-particles can be used as a vaccine delivery system, especially for Taiwan grouper iris virus infection (Grouper Iridovirus 〇f
Taiwan infectious disease)的防治。 【發明内容】 从太Γ此’本發明之目的在於㈣—種可助於包㈣學物質 、不只顆粒及其製造方法。其用來包埋的 :囊顆粒大小'可二到奈米，設備投資成本低，可==生 且操作技術間早，產品良率極高，所生產顆粒外觀 致0 ν 系統 本發明之另-目的’乃利用該奈米顆粒以作為疫苗的傳送 ’特別是應用於台灣石斑魚虹彩病毒❹症的防治。 200409656 根據本發明的目的，提出—種粒徑範圍約為2〇〜1〇〇〇⑽ 的：米顆粒’至少包括：-基質(base material)，該基質之 重里百分比約為30-99. 9%與一被包埋物（embedded substance)，其重量百分比約為〇. 17〇%。其中該基質係選自 由氫化植物油，氫化動物油及c8 Μ飽和脂肪酸及其衍生物所组 成之不水溶性基質族群中，或者是該基f係選自由二價離子成 膠類及其料物以及溫度變化成膠類及其衍生物所組成之水 溶性基質族群中。其巾該水溶性基f是為㈣酸納、果膠、洋 菜或石花菜，基質可包含甘油。該被包埋物是治療藥物，生理 活性物質，無機化合物’有機化合物，抗生素荷爾蒙，疫苗， 礦物質或維生素。 根據本發明的目的，提出一種製備粒徑範圍約為別〜 lOOOnm奈米顆粒的方法，包括步驟如下： > (a)熔融基貝，該基質之重量百分比約為加⑽.⑽，^ 2基質係選自由氫化植物油’氫化動物油及“飽和脂肪醇 ^衍生物所組成之不水溶性基f族群中4者是該基質係遥 X 一價離子成膠類及其魅物以及溫度變化成膠類及其衍 生物所組成之水溶性基質族群中； ㈦均勻混合該熔融之基質與—被包埋物；及 至一 輸管將該熔融基質與該被包埋物之混合物傳輸 5 ’該介f液體的溫度係低於㈣該基質的溫肩 並且該傳輸管連其傳送速度為至少約10升/小時， 丽。Μ連接有一噴嘴，該喷嘴口徑介於〇.〇1 mm至〇 8 根據本發明的另一 粒，至少包括：Taiwan infectious disease). [Summary of the Invention] From the point of view, the object of the present invention is to provide a substance, not only particles, and a method for manufacturing the same, which can assist in encapsulation. It is used for embedding: the size of the capsule particles can be two to nanometers, the equipment investment cost is low, it can == be born and the operation technology is early, the product yield is extremely high, and the appearance of the produced particles is 0 ν. -The purpose is to use the nano particles as a vaccine for delivery, especially for the prevention and treatment of Taiwan grouper iris virus rickets. 200409656 According to the purpose of the present invention, it is proposed that a kind of rice grains having a particle size in the range of about 20 ~ 100000⑽ includes at least:-a base material, and the percentage of the weight of the base is about 30-99. 9 17〇%。% and an embedded substance (Embedded substance), its weight percentage is about 171%. The matrix is selected from the group of water-insoluble matrixes composed of hydrogenated vegetable oil, hydrogenated animal oil, and c8 M saturated fatty acids and their derivatives, or the base f is selected from the group consisting of gels, materials, and temperatures from divalent ions. Into a water-soluble matrix group consisting of gums and their derivatives. The water-soluble group f is sodium gallate, pectin, agar, or stone flower, and the matrix may include glycerin. The embedded substance is a therapeutic drug, a physiologically active substance, an inorganic compound 'organic compound, an antibiotic hormone, a vaccine, a mineral or a vitamin. According to the purpose of the present invention, a method for preparing nano particles having a particle size ranging from about 100 nm to 100 nm is proposed, including the following steps: (a) Molten base, the weight percentage of the matrix is about ⑽.⑽, ^ 2 The matrix system is selected from the group consisting of hydrogenated vegetable oil, hydrogenated animal oil and "saturated fatty alcohol ^ derivatives, which are insoluble in the f group. The matrix system is X-valent ion-forming gels and their charms and gels with temperature changes. In a family of water-soluble matrixes composed of the same and their derivatives; ㈦ uniformly mixing the molten matrix and the embedded object; and transmitting a mixture of the molten matrix and the embedded object to a duct 5 'the media f The temperature of the liquid is lower than the warm shoulder of the substrate and the transfer tube and its transfer speed is at least about 10 liters / hour. The nozzle is connected to a nozzle with a diameter ranging from 0.01 mm to 0.08. Another capsule of the invention includes at least:
目的，提出一種包埋水產疫苗之奈米顆 該基質之重量百分比約為30_99.9%， 200409656 其中該基質係選自由二價離子成膠類及其衍生物以及溫度變 化成膠類及其衍生物所組成之水溶性基質族群中；及一水產疫 田作為被包埋物，其重量百分比約為〇•卜7〇%。其中該基質是 為海藻酸納、果膠、洋菜或石花菜，並且該水產疫苗係一台灣 石斑虹彩病毒的不活化疫苗。其製造過程中該基質與該水產疫 田係藉由約為150升/小時至300升/小時之傳輸速度，以一口 徑約為0·1 mm至0· 8 mm的噴嘴噴射造粒，製造出的奈米顆粒 之顆粒大小係介於2〇nm與1 OOOnm之間。 為讓本發明之上述目的、特徵、和優點能更明顯易懂，下 文特舉一較佳實施例，並配合所附圖式，作詳細說明如下： 【實施方式】 本發明係關於一種包埋化學物質之顆粒，特別是關於一種 包埋生理活性物質的顆粒。本發明的顆粒主要包括： (a) —基質（base material)，其重量百分比約為 30-99·9% ，及 (b) —被包埋物（embedded substance)，其重量百分比約 為 0.1 -70%。 應用於本發明之不水溶性基質，主要是一種或多種選自氫 化植物油、氫化動物油及^/或Cl"飽和脂肪酸及其衍生物的 物負。水》谷性基質係選自-種或多種的二價離子成膠類及其衍 生物所組成之族群中和溫度變化成膠類及其衍生物所組成2 族群中。其中，該二價離子成膠類及/或該溫度變化成膠類可 為海藻酸鈉、果膠、洋菜、石花菜。 氫化植物油主要是氫化椰子油、氫化棕櫚油、氫化大豆 油、氫化花生油或氫化蔬菜油。 氫化動物油主要是氫化牛油、氯化奶油、氯化魚油或氯化 9 200409656 豬油。 c 8-64飽和脂肪酸的主要特徵為在常溫下為固態，係下列 &任何-種或多種不限定數目之組合，例如：辛酸、壬酸、癸 +十駄、十一酸（月桂酸）、十三酸、十四酸（肉豆蔻酸）、 五酸、十六酸（棕櫚酸）、十七酸、十八酸（硬脂酸）、十九 酸、廿酸（花生酸）、廿一酸、以及廿二酸。 被包埋物為一化學物質，係選自治療藥物（如抗生素）、 t理活性物質（如荷爾蒙、疫苗等）或其它無機或有機化學成 分（如礦物質、維生素等）。 依據本發明的方法，傳輸溶融包埋物的速度，在10升/ 小日=以上皆可，而以150升八】、時至300升/小時之速度最佳。 ，鳴仏大或等於mm，而以〇·1 mm至〇·8 mm最佳。藉 著本毛明製造出的奈米顆粒粒徑範圍可由20 nm至1000 nm， 而以30 nm至180 nm為最多。 另外，應用於本發明中一種製造此顆粒的方法，包括步驟 如下： (a) 溶融基質； (b) 均勻混合基質及被包埋物； (c) 以一傳輸管將熔融的基質和被包埋物一起傳輸至一比 重靶圍約為〇·6 g/c«i3至1.5 g/cm3之介質液體中； 本lx明之方法的特徵為··介質液體的溫度至少低於熔融基質的 溫度5°C以上。 ' 一般而言，熔解基質的溫度约在10°C至20(TC之間，而以 40C至80 C為佳，視基質的種類而定。 再者’依據溶解基質的溫度而定，介質液體的溫度可控制 在約-10C至150°c之間，而以約2〇。〇至60°c為佳。 200409656 士依據本毛明的方法，傳輸溶融基質的速度，在1 〇升/小 、白可而以150升/小時至300升/小時之速度最佳。喷 嘴口 k大或等於〇·01 mm，而以〇1臟至〇·8咖最佳。 #本毛明方法產生顆粒的原理為：適當溫度的油相被注入介 二相中兩個不相溶相因注人力量與比重差異，使油相在介 貝液相中有如劇烈攪拌而產生大量裂解的微粒滴現象發生，當 大量分散裂解的微粒滴因本身的張力和介質液相的内聚力，使 微粒滴產生圓球型態，料適#的介該相溫度使微粒滴在產 生圓球型怨後’同時瞬間凝固形成固定的顆粒型態。 另外，應用於本發明中一較佳實施製造的奈米顆粒的裝 置，如第一圖所示，包括了構件如下： 一溶融基質102的容器（未顯示）； 一貯存被包埋物104的容器（未顯示）； 匕合基質102與被包埋物的容器101. 傳輸熔融基質102與被包埋物1〇4的傳輸管1〇5 :及 一貯存介質液體106的容器1〇7。 其中，傳輸管105連接有一驅動裝置1〇9,以驅動熔融的 基質102與被包埋物1〇4。此驅動裝置1〇9可為蠕動或齒輪泵 浦，而傳輸管1G5為-中空管柱，其和介質液體接觸部份接有 一喷嘴1U。中空管柱可為塑膠管、PEf、鐵弗龍管、銅管、 不錄鋼管等軟、硬管。喷嘴U1為中空管柱的延伸，材質可為 塑膠、PE、鐵弗龍、冑、不銹鋼等軟、硬材質；喷嘴口徑以 〇· 1 mm 至 〇· 8 mm 最佳。 在基質102之熔融物的溫度固定與介質液體1〇6溫度亦固 定的條件下，熔融基質102的傳輸速度愈快及噴嘴lu 口徑愈 小，則產生顆粒粒徑會愈小。 〜 200409656 基質102的脂肪酸與氫化動物、植物油對環境的溫度與酸 鹼值敏感度非常高，因此，需視被包埋物的不同而調整各種的 脂肪酸比例或動、植物油的氫化程度，以達到被包埋物1 可 容忍的基質熔融與凝固溫度。上述的基質丨〇2凝固後具有隔絕 空氣、水份及穩定性優異等特性，並在酸性環境中特別安定， 所以有攜帶被包埋物104通過人體胃部強酸環境的功能。根據 上述基質102的脂肪酸與氫化動、植物油特有的性質，本發明 製得的顆粒可以應用於如下所述的用途。 醫藥方面··因為脂肪酸化性穩定，有隔絕空氣與水份的特 性，可作為藥物的包覆儲存，並避免藥物氧化與潮解。另外， 月曰肪fee在Sst I*生環i兄中表現特別穩定，所以有攜帶被包埋物通過 人體胃酸的功能。其它方面還可以遮蔽口服藥劑的不適感，如 苦味。 食品方面：因為脂肪酸化性穩定，有隔絕空氣與水份的特 I*生可作為香料、原物料的包覆儲纟，並隔絕香料與原物料本 身氣味之散失，避免酸敗與潮解。由於所製成的顆粒外型美 觀’可增加食品附加價值。 尺畜產方面·因為脂肪酸化性穩定，有隔絕空氣與水我 的特性’可包埋保存疫苗、抗體與#養物f。在水產方面，逢 ㈣肪酸顆粒不溶解於水，並可調整顆粒比重。在包埋疫苗、 抗體《其他藥物後，顆粒可長時間存在於水的表面、懸浮或 沈降於底彳可有效餿食不同水域深度生活習性之魚類。此 卜在匕埋疫田、k體、或其他藥物之同時，亦可加入適當比 ”、、餌料α促進魚類吞食。也可以調整脂肪酸顆粒的熔 〜、 貝使匕們不被飼料調理所破壞，並順利到達畜產 12 200409656 動物的體内。 其它方面：可藉由調整脂肪酸顆粒的熔點，使得顆粒隨著 溫度變化而釋出碳粉與顏料，可利用於印刷產業。 以下實施例將進一步說明本發明，但必須了解的是這些實 施例的目的並不是要限制本發明的範圍。除非特別指明，以下 實例中的％是以重量計算，溫度為攝氏（它）。 實施例1 配方： 基質102 ··氫化硬棕油60%以及氫化椰子油3〇%; 被包埋物104 :台灣石斑虹彩病毒（TGIV)不活化疫苗 10〇/〇。 條件： 傳輸管105 :管徑20mm 流速：150升/小時 喷嘴 111 口徑：0. 8mm。 方法：把氫化硬棕油、氫化椰子油與TGIV不活化疫苗， 在55±3°C的溫度環境下，充分攪拌均勻，然後利用泵浦傳輸， 注入38±1°C水溶中造粒，而後送冷凍乾燥機乾燥。 結果·顆粒粒徑範圍為400-1000 nm，顆粒皆為圓球狀顆 粒，請參見第二圖。顆粒集結比重小於水，顆粒分散可懸浮於 水中，熔點為43°C，產量每一批次1〇〇〇公克。 效果：以此方式製備之福馬林及β—乙丙酸内酉旨 (β-propiolactone，BPL) TGIV 不活化疫苗（7Χ TCIDw/ml/fish)浸泡免疫石斑魚苗，15天後進行追加浸泡 13 200409656 (booster)，流水飼養 10 天後以 TGIV (l〇4 ° TCID5〇/ml/fish) 進行攻擊試驗，證明其免疫保護效果，請參見第三圖。 實施例2 配方： 基質102 :氫化硬棕油60%以及氫化椰子油30%; 被包埋物104 :台灣石斑虹彩病毒（TGIV)不活化疫苗 10〇/〇。 條件： 傳輸管105 :管徑20mm 流速：300升/小時 喷嘴 111 口徑：〇. 8mm。 方法··把氫化硬棕油、氫化椰子油與TGIV不活化疫苗， 在55±3°C的溫度環境下，充分攪拌均勻，然後利用泵浦傳輸， 注入38±1°C水溶中造粒，而後送冷凍乾燥機乾燥。 結果：顆粒粒徑範圍為30-180nm。 實施例3 配方：基質102 :氫化硬棕油1〇〇〇/0。 條件： 傳輸管105 :硬管管徑20mm， 流速：300升/小時， 噴嘴 111 口徑：0. 8mm。 方法：把氫化硬棕油在70±3°C的溫度環境下，充分攪拌 14 200409656 均勻’然後利用泵潘傳輸，注人45±Γ(：水溶中造粒，而後送冷 凍乾燥機乾燥。 ^ 結果：顆粒粒徑範圍為3〇_18〇 nm，請參見第四圖。顆粒 $結比重小於水，顆粒分散可懸浮於水中，熔點為53艺，產量 每一批次1000公克。 實施 配方：基質102 : 1%海藻酸鈉水溶液。 條件： 傳輸管105 :管徑20mm， 流速：300升/小時， 喷嘴口徑 111 : 〇.7mm。 方法：把1%海藻酸鈿水溶液在室溫環境下，充分攪拌均 勻，然後利用泵浦傳輸，注入室溫3%乳酸鈣水溶中造粒。 結果：可出現奈米顆粒，靜置一段時間奈米顆粒會集結在 水中出現下層雲霧狀。比重等於水，產量每一批次1〇〇〇公克。 效果：為水溶性奈米顆粒。 配方·基質102 : 4%Agar水溶液。 條件： 傳輸管105 :管徑20mm， 流速· 300升/小時， 噴嘴口徑：0. 7mm。 方法··把4%Agar水溶液在60°C環境下，充分攪拌均勻， 然後利用泵浦傳輸，注入室溫水溶中造粒。 15 409656 。果可出現奈米顆粒，靜置一段時間奈米顆粒會集結在 7中出現上層雲霧狀。比重等於水，產量每一批次1〇〇〇公克。 效果·為水溶性奈米顆粒。 與習知堆積作用產顆粒和喷霧懸浮流 動床產生顆粒相較，本發明之生產顆粒除了具有優異的穩定 性，可穩定包覆特別是生理物質之外的化學物質，可藉由調整 顆粒熔點與比重以配合應用領域之需要，並可製造出品質穩定 的奈米顆粒。另外，習知堆積作用生產顆粒的最大缺點為操作 技術複雜與不能連續生產，喷霧懸浮流動床生產顆粒的最大缺 點為設備成本高、產量低與產品不良率極高，而且此兩大方法 生產過程均非常耗時。但本發明生產奈米顆粒的方法均可-- 克服這些困難。 依照本發明之方法製造出之奈米顆粒可以被廣泛的應用 在醫藥、食品上。以下以實驗證明本發明之奈米顆粒可以成功 的應用在台灣石斑魚虹彩病毒感染症的防治。 材料方法 一、 石斑虹彩病毒不活化疫苗之大量製備 將TGIV以Μ01 = 0· 1〜1接種至單層培養之石斑魚泳鰾細胞 株（swimbladder，SB cell line)，於 25°C 下培養 7〜14 天後收 集病毒液，經重複結凍解凍，以福馬林及BPL分別於4、20°C 下作用4天，經透析後即可使用。 二、 比較有無包埋之石斑虹彩病毒不活化疫苗以浸泡法免 疫石斑魚之效果差異 1、將製備好的石斑虹彩病毒不活化疫苗以海水稀釋後， 16 200409656 對石斑魚苗浸泡免疫1小時，以流水飼養15天後進行追加浸 泡1小時，移入海水飼養10天（A組）。或浸泡免疫1小時後流 水飼養25天（B組），後以104 GTCID5〇/ml TGIV進行攻擊試驗， 觀察兩週記錄死亡率。 2、將所製備好之虹彩病毒不活化疫苗以根據實施例2製 造出的奈米顆粒包埋後，以海水稀釋對石斑魚苗進行浸泡免疫 1】、日^ ’以流水飼養15天後進行追加浸泡1小時，移入海水飼 養丨〇天（A組）。或浸泡免疫1小時後流水飼養25天（B組）， 後以1〇4 °TCID5〇/ml TGIV進行攻擊試驗，觀察兩週記錄死亡率。Aim: To propose a nano vaccine for embedding aquatic vaccine. The weight percentage of the matrix is about 30-99.9%, 200409656. The matrix is selected from the group consisting of gels and derivatives derived from divalent ions and gels and derivatives derived from temperature change. In a water-soluble matrix group composed of the objects; and a fish epidemic field as an embedded object, the weight percentage of which is about 70%. Wherein the substrate is sodium alginate, pectin, amaranth or stone broccoli, and the aquatic vaccine is a Taiwan grouper iridescent virus inactivated vaccine. In the manufacturing process, the substrate and the aquatic epidemic field are granulated by a spraying nozzle with a diameter of about 0.1 mm to 0.8 mm at a transfer speed of about 150 liters / hour to 300 liters / hour. The particle size of the nano particles is between 20nm and 1000nm. In order to make the above-mentioned objects, features, and advantages of the present invention more comprehensible, a preferred embodiment is given below in conjunction with the accompanying drawings for detailed description as follows: [Embodiment] The present invention relates to an embedding method. Particles of a chemical substance, in particular, a particle in which a physiologically active substance is embedded. The particles of the present invention mainly include: (a)-a base material, whose weight percentage is about 30-99.9%, and (b)-an embedded substance, whose weight percentage is about 0.1- 70%. The water-insoluble substrate used in the present invention is mainly one or more kinds selected from hydrogenated vegetable oils, hydrogenated animal oils, and / or Cl " saturated fatty acids and derivatives thereof. The "water" cereal matrix is selected from the group consisting of one or more divalent ion-forming gels and their derivatives and the temperature-changing gels and their derivatives. The divalent ion-forming gels and / or the temperature-changing gels may be sodium alginate, pectin, agar, and cauliflower. The hydrogenated vegetable oils are mainly hydrogenated coconut oil, hydrogenated palm oil, hydrogenated soybean oil, hydrogenated peanut oil or hydrogenated vegetable oil. Hydrogenated animal oils are mainly hydrogenated tallow, chlorinated butter, chlorinated fish oil or chlorinated 9 200409656 lard. The main characteristic of c 8-64 saturated fatty acid is that it is solid at normal temperature, and it is the following & any or a combination of an unlimited number of, for example: octanoic acid, nonanoic acid, decyl + decadecyl, undecanoic acid (lauric acid) , Tridecanoic acid, myristic acid (myristic acid), pentaic acid, hexadecanoic acid (palmitic acid), heptadecanoic acid, octadecanoic acid (stearic acid), undecanoic acid, osmic acid (arachidic acid), 廿Monoacid, and adipic acid. The embedded substance is a chemical substance, which is selected from therapeutic drugs (such as antibiotics), physioactive substances (such as hormones, vaccines, etc.) or other inorganic or organic chemical components (such as minerals, vitamins, etc.). According to the method of the present invention, the speed of transferring the melted embedment is 10 liters / day = more than the above, and the speed of 150 liters and 300 liters per hour is optimal. , Ming is large or equal to mm, and the best is 0.1 mm to 0.8 mm. Nano-particles manufactured by Ben Maoming range in particle size from 20 nm to 1000 nm, with 30 nm to 180 nm being the largest. In addition, a method for manufacturing the particles used in the present invention includes the following steps: (a) melting the matrix; (b) uniformly mixing the matrix and the embedding substance; (c) using a transfer tube to melt the matrix and the coating substance The burial is transferred together into a medium liquid with a specific gravity target around 0.6 g / c «i3 to 1.5 g / cm3; the method of this lxming method is characterized by the temperature of the medium liquid being at least lower than the temperature of the molten matrix 5 Above ° C. 'Generally speaking, the temperature of the melting matrix is about 10 ° C to 20 ° C, and preferably 40C to 80 ° C, depending on the type of matrix. Furthermore,' depending on the temperature of the dissolving matrix, the medium liquid The temperature can be controlled between about -10C to 150 ° C, and preferably about 20.0 to 60 ° C. 200409656 According to the method of Ben Maoming, the speed of transferring the melting matrix is 10 liters / hour White and white are the best at a speed of 150 liters / hour to 300 liters / hour. The nozzle opening k is larger than or equal to 0.01 mm, and the best is from 0 to 0. 8 coffee. # 本 毛 明 Method produces particles The principle is: the oil phase at the appropriate temperature is injected into the two immiscible phases in the mesophase. Due to the difference in strength and specific gravity, the oil phase in the mesophase has a large number of cracked particulate droplets as if vigorously stirred. When a large number of dispersed and broken particles drop due to their own tension and the cohesion of the liquid phase of the medium, the particles form a spherical shape. The phase temperature of the material makes the particles instantly solidify at the same time after the spherical shape is generated. Forms a fixed particle shape. In addition, it is used in a preferred embodiment of the present invention. The granular device, as shown in the first figure, includes the following components: a container (not shown) that melts the substrate 102; a container (not shown) that stores the embedded object 104; and the substrate 102 and the embedded object The container 101. The transfer tube 102 for transferring the molten matrix 102 and the embedded object 104 is a container 10 for storing the liquid 106. Among them, the transfer tube 105 is connected with a driving device 109 for driving. The melted matrix 102 and the embedded object 104. The driving device 109 may be a peristaltic or gear pump, and the transmission tube 1G5 is a hollow tube string, and a nozzle 1U is connected to the medium contact portion. The hollow pipe string can be plastic, PEf, Teflon pipe, copper pipe, non-recording steel pipe and other soft and hard pipes. The nozzle U1 is an extension of the hollow pipe string, and the material can be plastic, PE, Teflon, 胄Soft and hard materials, such as stainless steel, stainless steel; nozzle diameter is best from 0.1 mm to 0.8 mm. Under the condition that the temperature of the melt of the substrate 102 is fixed and the temperature of the medium liquid 106 is also fixed, the molten substrate 102 is transported. The faster the speed and the smaller the diameter of the nozzle lu, the smaller the particle size will be. ~ 20 0409656 The fatty acids and hydrogenated animal and vegetable oils of the substrate 102 are very sensitive to the environmental temperature and pH value. Therefore, the proportion of various fatty acids or the degree of hydrogenation of animal and vegetable oils needs to be adjusted according to the different embedded materials to achieve the Embedment 1 Tolerable matrix melting and solidification temperature. The above-mentioned matrix 丨 02 has the characteristics of excellent insulation from air, moisture and stability after being solidified, and is particularly stable in acidic environments, so it has an embedded object 104 Through the function of the strong acid environment of the human stomach. According to the properties specific to the fatty acids and hydrogenated animal and vegetable oils of the matrix 102 described above, the granules prepared by the present invention can be applied to the applications described below. In terms of medicine ... Because the fatty acid is stable and has the characteristics of blocking air and moisture, it can be used as a coating to store the drug and avoid drug oxidation and deliquescent. In addition, Yuefu is particularly stable in Sst I * ring, so it has the function of carrying embedded matter through the human stomach acid. Other aspects can also mask the discomfort of oral medications, such as bitterness. Food: Because of its stable fatty acid properties, it can be used as a coating and storage for spices and raw materials to isolate air and water, and to isolate the loss of flavor from the raw materials to avoid rancidity and deliquescent. Due to the beautiful appearance of the granules, the added value of food can be increased. In terms of animal production, because it has stable fatty acid properties, it has the property of blocking air and water. It can embed and store vaccines, antibodies, and nutrients. In terms of aquatic products, every fatty acid particle is insoluble in water, and the particle specific gravity can be adjusted. After embedding vaccines, antibodies, and other drugs, particles can exist on the surface of water for a long time, suspended or settled on the bottom, and can effectively eat fish of different depths of life. At the same time as burying the epidemic field, K body, or other drugs, you can also add appropriate ratios ", bait α to promote fish swallowing. You can also adjust the melting of fatty acid particles ~, so that the daggers are not damaged by feed conditioning. And successfully reached the animal's body 12 200409656. Other aspects: The melting point of fatty acid particles can be adjusted so that the particles release carbon powder and pigment with temperature changes, which can be used in the printing industry. The following examples will further explain The present invention, but it must be understood that the purpose of these examples is not to limit the scope of the present invention. Unless otherwise specified, the% in the following examples is calculated by weight and the temperature is Celsius (it). Example 1 Formulation: Matrix 102 · Hydrogenated hard palm oil 60% and hydrogenated coconut oil 30%; Embedment 104: Taiwan grouper iridescent virus (TGIV) inactivated vaccine 10 / 〇 Conditions: Transfer tube 105: 20mm diameter Flow rate: 150 Liter / hour nozzle 111 caliber: 0.8mm. Method: The hydrogenated hard palm oil, hydrogenated coconut oil and TGIV inactivated vaccine are stirred well at a temperature of 55 ± 3 ° C. It is pumped and pumped, injected into 38 ± 1 ° C water solution for granulation, and then sent to a freeze dryer for drying. Results · The particle size range is 400-1000 nm, and the particles are all spherical particles, see the second figure. Particles The aggregate specific gravity is less than water, the particles can be dispersed in water, the melting point is 43 ° C, and the yield is 1000 grams per batch. Effect: The formalin and β-ethylpropionate prepared by this method (β- Propiolactone (BPL) TGIV inactivated vaccine (7X TCIDw / ml / fish) was immersed in grouper fry. 15 days later, additional immersion 13 200409656 (booster), and 10 days after running in water, TGIV (104 ° TCID50 / ml /) fish) to conduct an attack test to prove its immune protection effect, please refer to the third figure. Example 2 Formulation: Matrix 102: hydrogenated hard palm oil 60% and hydrogenated coconut oil 30%; embedment 104: Taiwan grouper iridescent virus (TGIV) Inactivated vaccine 10 / 〇 Conditions: Transfer tube 105: 20mm diameter Flow rate: 300 liters / hour Nozzle 111 Caliber: 0.8mm. Method ·· Hydrogenated hard palm oil, hydrogenated coconut oil, and TGIV are not activated Vaccine, fully under the temperature environment of 55 ± 3 ° C Stir well, then transfer it by pump, inject it into 38 ± 1 ° C water solution for granulation, and then send it to freeze dryer for drying. Result: The particle size range is 30-180nm. Example 3 Formulation: Matrix 102: hydrogenated hard palm oil 〇〇〇〇 / 〇。 Conditions: transmission tube 105: hard tube diameter 20mm, flow rate: 300 liters / hour, nozzle 111 diameter: 0.8mm. Method: hydrogenated hard palm oil at 70 ± 3 ° C temperature environment Next, fully stir 14 200409656 evenly, then use pump pan to convey, inject 45 ± Γ (: granulate in water solution, and then send to freeze dryer to dry. ^ Result: The particle size ranges from 30 to 18 nm, see figure 4. Granules The knot specific gravity is smaller than water. The particles can be suspended in water with a melting point of 53 g. The yield is 1000 grams per batch. Implementation formula: Matrix 102: 1% aqueous sodium alginate solution. Conditions: transmission tube 105: tube diameter 20mm, flow rate: 300 liters / hour, nozzle diameter 111: 0.7mm. Method: Agitate 1% alginate solution in water at room temperature, stir well, then transfer by pump, and inject 3% calcium lactate in water at room temperature to granulate. Result: Nano particles can appear, and the nano particles will gather in the water for a period of time and form a lower cloud shape. The specific gravity is equal to water and the yield is 1,000 g per batch. Effect: It is water-soluble nano particles. Formulation Base 102: 4% Agar water solution. Conditions: Transmission tube 105: 20mm diameter, flow rate 300 liters / hour, nozzle diameter: 0.7mm. Method · Stir 4% Agar aqueous solution in 60 ° C environment, stir well, then transfer it by pump and inject it into room temperature water solution to granulate. 15 409656. If nano-particles can appear, the nano-particles will gather in 7 for a period of time and appear as upper clouds. The specific gravity is equal to water and the yield is 1,000 g per batch. Effect · It is water-soluble nano particles. Compared with the conventional production of particles produced by stacking and particles produced by spray suspension fluidized bed, in addition to the excellent stability of the particles produced by the present invention, it can stably coat chemical substances other than physiological substances, and the particle melting point can be adjusted by With the specific gravity to meet the needs of the application field, and can produce nano particles with stable quality. In addition, the biggest shortcomings of the conventional production of granules are the complex operation technology and continuous production. The biggest shortcomings of the production of granules in the spray suspension fluidized bed are high equipment cost, low output and extremely high product failure rate. The processes are very time consuming. However, the method for producing nano particles according to the present invention can all overcome these difficulties. The nano particles produced according to the method of the present invention can be widely used in medicine and food. The following experiments show that the nano particles of the present invention can be successfully applied to the prevention and treatment of Taiwan grouper iris virus infection. Material Method I. Large-scale preparation of grouper iridescent virus inactivated vaccine. TGIV was inoculated to monolayer cultured grouper swimmer cell line (swimbladder, SB cell line) at M01 = 0.1 to 1 and cultured at 25 ° C. 7 The virus solution was collected after ~ 14 days, and then thawed repeatedly. Formalin and BPL were used at 4 and 20 ° C for 4 days. They were ready for use after dialysis. Comparison of the effect of immunization of grouper with or without embedded grouper iridescent virus inactivated vaccine 1. Immerse the prepared grouper iridescent virus inactivated vaccine with seawater. 16 200409656 Immunize grouper fry for 1 hour. After 15 days of feeding in running water, an additional immersion was performed for 1 hour, and the animals were transferred to seawater for 10 days (group A). Or immunized for 1 hour and soaked in water for 25 days (group B), then challenged with 104 GTCID50 / ml TGIV, and observed the mortality for two weeks. 2. After the prepared iridescent virus inactivated vaccine is embedded with the nano particles manufactured according to Example 2, the grouper fry is soaked and immunized by diluting with seawater. Soak for 1 hour and move to seawater for 0 days (group A). Or immunized for 1 hour and immersed in flowing water for 25 days (group B), and then challenged with 104 ° TCID50 / ml TGIV, and observed the mortality for two weeks.
Hi 、石斑虹彩病毒不活化疫苗之大量製備 培養病毒後製造不活化疫苗以供實驗所需。 一、比較有無包埋之石斑虹彩病毒不活化疫苗以浸泡法免 疫石斑魚之效果差異 如第五圖所示，以有包埋及無包埋福馬林和BpL製備TGIV 不/舌化疫备（7χ 1〇 TCIDso/ml)浸泡免疫石斑魚（平均體長 2.3cm，體重約 i.8g)後，以 TGIV (1〇4。TCIDs()/fish)腹腔注 射進行攻擊試驗的結果。黑色實心賴㈣為未免疫組，^攻 =實驗進行後10天，檢體已全部死亡，累積存活率為〇%。疫 :處理組中’實〜、表示經奈米膠囊包埋之疫苗處理組；空心表 示未經奈米膠囊包埋之疫苗處理組。卩BpL所製備之奈米膠囊 疫苗，經追加浸泡後其累.積存活率為9〇%(黃色實心三角妒）. 未= fBPL製備不活化疫苗經追加浸泡後其累積存^率為 〇( η色二角形空心）。以褐馬林所製備之不活化疫苗其保護 17Hi, mass preparation of grouper iridescent virus inactivated vaccine After incubating the virus, an inactivated vaccine is produced for experimental needs. I. Comparison of the effect of immunization of grouper with or without embedded grouper iridescent virus inactivated vaccine by group immersion as shown in the fifth figure. Preparation of TGIV non / tongue epidemic preparation with or without embedded formalin and BpL ( 7χ 10TCIDso / ml) was immersed in grouper (average body length: 2.3cm, body weight: approximately i.8g), and then challenged with TGIV (104.TCIDs () / fish) by intraperitoneal injection. The black solid Lai Lai is the non-immunized group. ^ Attack = 10 days after the experiment, all the specimens have died, and the cumulative survival rate is 0%. Epidemic: In the treatment group, ‘actually’ means vaccine treatment group embedded with nanocapsules; hollow indicates vaccine treatment group not embedded with nanocapsules.奈 The nanocapsule vaccine prepared by BpL is cumulative after additional soaking. The cumulative survival rate is 90% (yellow solid triangle jealousy). Not = the cumulative storage rate of the inactivated vaccine prepared by fBPL after additional soaking is 0 ( η-colored dihedral hollow). Protection of inactivated vaccines prepared from brown marlin 17
OJO OJO 效果均比BPL所製 埋）〇 傷之疫苗效果差（不論有無經奈米膠囊包 將石斑虹彩病毒不活化疫苗愈 反田與奈未膠囊包埋技術結合 斤製成的不米膠囊疫苗初步的結果看來對石斑〜 木症的防4實有保護效果。將疫苗製成奈米顆粒 ^免疫，免疫25天後（經過追加浸泡）其保護效果達9⑽但 未經過膠囊化的疫苗其保護效果達·，由此証明疫苗膠囊化-確實可增加此疫苗對魚類的保護效果。 ,在本實驗中证明了利用本發明包埋技術所製造之奈米顆 粒確貝可使石斑魚對此疾病產生免疫，且經過膠囊化之疫苗轉 實比未經膠囊化的疫苗還有效。 綜上所述，雖然本發明已以一較佳實施例揭露如上，然其 並非用以限定本發明，任何熟習此技藝者，在不脫離本發明之 精神和範圍内，當可作各種之更動與潤飾，因此本發明之保護 範圍當視後附之申請專利範圍所界定者為準。 200409656 【圖式簡單說明】 第一圖顯不本發明中一較佳實施製造的奈米顆粒的裝置。 第二圖顯示依照本發明第一實施例製造的奈米顆粒圓球 狀顆粒的SEM圖。 第三圖繪不依照本發明第一實施例製造的奈米疫苗對石 斑魚苗之免疫保護效果評估。以福馬林和BPL製備之tgiv不 活化疫苗（7x 102 TCID5G/ml/fish)浸泡免疫石斑魚，15天後 進行追加浸泡，流水飼養十天後，以TGIV (1〇4.QTCID5Q/ml/fish) 腹腔/主射進行攻擊试驗的結果。顯示之死亡率二重複之平均 值。 第四圖繪示依照本發明第三實施例製造的奈米顆粒顆粒 大小分佈圖。 第五圖顯示以有包埋及無包埋福馬林和Bpl製備tg IV不 活化疫苗（7x 1〇2 TCH/ml)浸泡免疫石斑魚後，以tgiv (104G TCID5〇/fish)腹腔注射進行攻擊試驗的結果。死亡率為 二重複之平均值。 19OJO OJO effect is lower than that produced by BPL) 0 The vaccine of the wound is ineffective (regardless of the presence or absence of nano capsules, the grouper iridescent virus inactivated vaccine Yufantian and nano-capsule embedding technology combined with the Jin capsule capsule vaccine made preliminary The result seems to have a protective effect on the prevention of sclerotinia spp. ~ 4. The vaccine is made into nano granules ^ immunized. After 25 days of immunization (after additional soaking), the protective effect is 9⑽, but the vaccine has not been encapsulated. The protective effect is up to, which proves that the vaccine is encapsulated-it can indeed increase the protective effect of the vaccine on fish. In this experiment, it was proved that the nano-particles made from the embedding technology of the present invention can make grouper to this disease. Generate immunity, and the encapsulated vaccine is more effective than the unencapsulated vaccine. In summary, although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the present invention, any familiarity Those skilled in the art can make various modifications and retouching without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be defined by the scope of the attached patent application as follows: 200409656 [Brief description of the diagram] The first diagram shows a device for manufacturing nano particles according to a preferred embodiment of the present invention. The second diagram shows the SEM of the spherical nano particles produced according to the first embodiment of the present invention. Figure 3. The third figure shows the evaluation of the immunoprotective effect of the nano vaccine produced in accordance with the first embodiment of the present invention on grouper fry. The tgiv inactivated vaccine (7x 102 TCID5G / ml / fish) prepared with formalin and BPL was immersed for immunization. Grouper, after 15 days of additional immersion, after 10 days of running in water, the results of the challenge test with TGIV (104.QTCID5Q / ml / fish) abdominal cavity / main shot. The average of two replicates of the mortality rate shown. The four figures show the size distribution of nano particles produced according to the third embodiment of the present invention. The fifth figure shows the preparation of tg IV inactivated vaccine (7 x 10 2 TCH / with and without embedded formalin and Bpl). ml) Immunized grouper, immersed in tgiv (104G TCID50 / fish) intraperitoneally to conduct challenge test results. Mortality is the average of two replicates. 19