TW202421775A - Composition and Method of Treatment for Cell Growth Using Antler Stem Cell-Conditioned Medium - Google Patents
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Abstract
Description
本發明有關於一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法。 The present invention relates to a composition and method for treating cell growth using antler stem cell-conditioned medium.
已知鹿茸一旦從其角柄上脫落會因基於幹細胞的過程而完全再生;特定地,先前完成的研究已提供證據指出鹿茸幹細胞不僅完全生成鹿茸,而且亦促進鹿茸皮膚傷口的完美癒合;因此,需要利用由鹿茸幹細胞產生並與之相關的這些化合物應用於人體以促進細胞生長及癒合。 It is known that antler velvet, once shed from its horn, completely regenerates due to a stem cell-based process; specifically, previously completed studies have provided evidence that antler stem cells not only completely generate antler velvet, but also promote the perfect healing of antler skin wounds; therefore, there is a need to utilize these compounds produced by and associated with antler stem cells for application in the human body to promote cell growth and healing.
因此,申請人得開發出本發明案創作,並提出申請。其中 Therefore, the applicant must develop the invention and file an application.
本發明之目的,在提供一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其細胞生長組合 物,包含封裝在奈米顆粒中之鹿茸幹細胞-條件培養基(ASC-CM)。 The purpose of the present invention is to provide a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the cell growth composition comprises antler stem cell-conditioned medium (ASC-CM) encapsulated in nanoparticles.
本發明之目的,在提供一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其細胞生長組合物,包含由達爾伯克改良伊格爾培養基(DMEM)、胎牛血清(FBS)及鏈絲菌素中培養之鹿茸幹細胞得到之鹿茸幹細胞條件培養基(ASC-CM)。 The purpose of the present invention is to provide a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the cell growth composition comprises antler stem cell conditioned medium (ASC-CM) obtained from antler stem cells cultured in Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and streptomycin.
本發明之目的,在提供一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其細胞生長組合物,包括上述細胞生長組合物之一的給藥步驟。 The purpose of the present invention is to provide a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, wherein the cell growth composition includes a drug administration step of one of the above-mentioned cell growth compositions.
圖1:係以示意圖描繪在鹿茸幹細胞-條件培養基(ASC-CM)中細胞生長的實驗程序。 Figure 1: Schematic diagram of the experimental procedure for cell growth in antler stem cell-conditioned medium (ASC-CM).
圖2:係以示意圖描繪ASC-CM的製備過程。 Figure 2: A schematic diagram depicting the preparation process of ASC-CM.
圖3:描繪使用ASC-CM的MDCK細胞(麥登-達比犬類腎臟上皮細胞)生長的實驗結果。 Figure 3: Depicting the results of an experiment using ASC-CM to grow MDCK cells (Madden-Darby canine renal epithelial cells).
圖4:描繪顯微鏡下的MDCK細胞生長結果。 Figure 4: Depicts the MDCK cell growth results under a microscope.
圖5:描繪使用ASC-CM的NIH-3T3(鼠胚胎纖維母細胞系)細胞生長的實驗結果。 Figure 5: Depicts the results of an experiment using ASC-CM to grow NIH-3T3 (mouse embryonic fibroblast cell line).
圖6:描繪顯微鏡下的NIH-3T3細胞生長結果。 Figure 6: Depicts the NIH-3T3 cell growth results under a microscope.
圖7:係以示意圖描繪封裝在奈米顆粒中的ASC-CM的製備方法實施例。 Figure 7: A schematic diagram illustrating an embodiment of a method for preparing ASC-CM encapsulated in nanoparticles.
圖8:列出ASC-CM奈米顆粒的特徵。 Figure 8: List the characteristics of ASC-CM nanoparticles.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其組合物可包括本發明在本文所述基本元素及限制以及本文所述任何附加或可選的成分、組分或限制,該等組合物是由以上項目組成或基本上由其組成。 The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition may include the basic elements and limitations of the present invention described herein and any additional or optional ingredients, components or limitations described herein, and the composition is composed of or substantially composed of the above items.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,在本說明書及後附申請專利範圍中使用的單數形式“a”、“an”及“the”亦包括複數引用(除非上下文另有明確規定),例如“一個”細胞一詞包括複數個細胞(包括有其混合物)。 The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The singular forms "a", "an" and "the" used in this specification and the attached patent application also include plural references (unless the context clearly stipulates otherwise). For example, the term "a" cell includes plural cells (including mixtures thereof).
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,在量值的上下文中,“大約”是指基於所述值的平均偏差最大±20%,較佳±10%,或更佳±5%;例如相對於總脂質/兩親體的莫耳濃度,約30莫耳%的陰離子脂質的量是指30莫耳%±6莫耳%的陰離子脂質,較佳30莫 耳%±3莫耳%,或更佳30莫耳%±1.5莫耳%。 The present invention discloses a composition and method for treating cell growth using velvet antler stem cell-conditioned medium. In the context of a quantity value, "approximately" means a maximum deviation of ±20%, preferably ±10%, or more preferably ±5% based on the average deviation of the value; for example, relative to the molar concentration of total lipid/amphiphile, an amount of about 30 mol% anionic lipid means 30 mol% ±6 mol% anionic lipid, preferably 30 mol% ±3 mol%, or more preferably 30 mol% ±1.5 mol%.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其“脂質”是指其作為一般用詞的傳統意義,包括脂肪、脂質及原生質的醇-醚可溶成分(其不溶於水);脂質是由脂肪、脂肪油、精油、蠟、類固醇、固醇、磷脂、醣脂、硫脂、胺脂質、色脂及脂肪酸組成,該用詞包括天然存在的脂質及合成脂質兩者;與本發明相關的較佳脂質是:類固醇及固醇,尤其是膽固醇、磷脂,包括有磷脂醯及磷脂醯膽鹼及磷脂醯乙醇胺,以及神經鞘磷脂;只要存在脂肪酸,其長度可能約為12至24個碳鏈,含有高達6個雙鍵;脂肪酸是連接到主鏈,主鏈可衍生自甘油;一種脂質內的脂肪酸可不同(不對稱),或可能只存在一個脂肪酸鏈,例如溶血卵磷脂;亦可能是混合配方,尤其當非陽離子脂質是從天然來源得到時,天然來源如由蛋黃、牛心臟、腦或肝臟或大豆中純化的卵磷脂(磷脂醯膽鹼)。 The present invention is a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, wherein "lipid" refers to its traditional meaning as a general term, including fat, lipids and alcohol-ether soluble components of protoplasm (which are insoluble in water); lipids are composed of fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, carbohydrates, sulfolipids, amine lipids, chromophores and fatty acids, and the term includes both naturally occurring lipids and synthetic lipids; preferred lipids related to the present invention are: steroids and sterols, especially cholesterol, phospholipids, including phospholipids Acyl and phosphatidylcholine and phosphatidylethanolamine, as well as sphingomyelin; fatty acids, when present, may be about 12 to 24 carbon chains in length, with up to 6 double bonds; fatty acids are attached to a backbone chain, which may be derived from glycerol; fatty acids within a lipid may be different (asymmetric), or only one fatty acid chain may be present, such as lysolecithin; mixed formulations may also be present, especially when the non-cationic lipid is obtained from a natural source, such as purified lecithin (phosphatidylcholine) from egg yolk, beef heart, brain or liver, or soy.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其“有效量”是足以產生有益或期望結果的量;有效量可在一次或多次給藥、使用或劑量中給藥。 The present invention provides a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the "effective amount" is an amount sufficient to produce a beneficial or desired result; the effective amount can be administered in one or more administrations, uses or dosages.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞 生長之組合物及方法,其“受試者”、“個體”或“患者”在本文中可互換使用,其是指脊椎動物,較佳是哺乳動物,更佳是人類;哺乳動物包括(但不限於)鼠類、猿猴、人類、農場動物、運動動物及寵物。 The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The terms "subject", "individual" or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, and more preferably humans; mammals include (but are not limited to) mice, monkeys, humans, farm animals, sports animals and pets.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其鹿茸是唯一已知當從其角柄上脫落時可完全再生的哺乳動物器官,而且已知傷口會癒合而不留下疤痕;下面的範例描述ASC-CM對細胞生長的治療作用,說明ASC-CM是由多種促進人體細胞生長的化合物所組成。 The present invention is a composition and method for treating cell growth using antler stem cell-conditioned medium. Antler is the only known mammalian organ that can completely regenerate when it falls off its horn stalk, and it is known that wounds will heal without leaving scars. The following example describes the therapeutic effect of ASC-CM on cell growth, indicating that ASC-CM is composed of multiple compounds that promote human cell growth.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其組合物包括封裝在奈米顆粒如微脂粒或脂質奈米顆粒(LNP)內的ASC-CM,其可用口服、外用或注射方式給藥;在另一實施例中,本發明的ASC-CM組合物包括封裝有ASC-CM的脂質微胞;在又一實施例中,本發明的ASC-CM組合物包括封裝有ASC-CM的微脂粒。 The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition comprises ASC-CM encapsulated in nanoparticles such as vesicles or lipid nanoparticles (LNPs), which can be administered orally, topically or by injection; in another embodiment, the ASC-CM composition of the present invention comprises lipid micelles encapsulating ASC-CM; in yet another embodiment, the ASC-CM composition of the present invention comprises vesicles encapsulating ASC-CM.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其組合物包括封裝在上述奈米顆粒中的冷凍乾燥形式ASC-CM,可將冷凍乾燥的ASC-CM奈米顆粒復原以用於注射給藥;在一實施例中,每個奈米顆粒 的尺寸在約100奈米至約10000奈米之間、約250奈米至約7500奈米之間,或約500奈米至約5000奈米之間;本發明的組合物尚包括賦形劑或佐劑,可能的賦形劑範例包括硫酸鋁鈉或蔗糖。 The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium, wherein the composition includes a freeze-dried form of ASC-CM encapsulated in the above-mentioned nanoparticles, and the freeze-dried ASC-CM nanoparticles can be reconstituted for injection; in one embodiment, the size of each nanoparticle is between about 100 nanometers and about 10,000 nanometers, between about 250 nanometers and about 7,500 nanometers, or between about 500 nanometers and about 5,000 nanometers; the composition of the present invention also includes a shaping agent or adjuvant, and possible shaping agent examples include sodium aluminum sulfate or sucrose.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,亦可與一或多種藥學上可接受的賦形劑或佐劑結合,將本發明的ASC-CM組合物以封裝有ASC-CM的膠囊如凝膠蓋形式配製以用於口服給藥;在一實施例中,本發明的細胞生長組合物包括本文所揭示奈米顆粒ASC-CM的任何實施例,奈米顆粒ASC-CM是封裝在膠囊如凝膠蓋以用於口服給藥;在另一實施例中,本發明的細胞生長組合物包括本文所揭示冷凍乾燥粉末形式的ASC-CM奈米顆粒的任何實施例,該粉末是封裝在膠囊中以用於口服給藥,此一製劑的實施例將在以下實施例中揭露。 The present invention discloses a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, which can also be combined with one or more pharmaceutically acceptable excipients or adjuvants to prepare the ASC-CM composition of the present invention in the form of a capsule encapsulated with ASC-CM, such as a gel cap, for oral administration. In one embodiment, the cell growth composition of the present invention includes the nanoparticle AS disclosed herein. In any embodiment of ASC-CM, the nanoparticles ASC-CM are encapsulated in a capsule such as a gel cap for oral administration; in another embodiment, the cell growth composition of the present invention includes any embodiment of ASC-CM nanoparticles in the form of a freeze-dried powder disclosed herein, which is encapsulated in a capsule for oral administration. An embodiment of such a preparation will be disclosed in the following embodiments.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,亦可將本發明的組合物配製以用於外用給藥,在一實施例中,其組合物包括以上所揭示任何形式的ASC-CM組合物及藥學上可接受的賦形劑或佐劑,將其封裝在可用手破壞的管子中,該管子可輕易用手破壞使ASC-CM釋出以用於外用給藥。 The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The composition of the present invention can also be formulated for external administration. In one embodiment, the composition includes any form of ASC-CM composition disclosed above and a pharmaceutically acceptable excipient or adjuvant, which is packaged in a manually destructible tube, which can be easily destructed by hand to release ASC-CM for external administration.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,針對本發明的一或數個方面討論的所有較佳實施例亦相關所有其他方面;這尤其是指中性或陰離子脂質的數量及類型、活性劑的數量及類型、用於聯合治療的進一步活性劑的數量及類型,以及待治療疾病的類型。 The present invention relates to a composition and method for treating cell growth using antler stem cell-conditioned medium. All preferred embodiments discussed for one or more aspects of the present invention also relate to all other aspects; this particularly refers to the amount and type of neutral or anionic lipids, the amount and type of active agents, the amount and type of further active agents used in combination therapy, and the type of disease to be treated.
本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,以下實施例應僅為舉例說明,並非用以限制本發明的範圍,其他通用及特定配置對熟諳此藝者將顯而易見。 The present invention discloses a composition and method for treating cell growth using antler stem cell-conditioned medium. The following embodiments are intended to be illustrative only and are not intended to limit the scope of the present invention. Other general and specific configurations will be apparent to those skilled in the art.
首先,敬請配合參閱如圖一至圖八及以下詳細說明所示:本發明一種使用鹿茸幹細胞-條件培養基治療細胞生長之組合物及方法,其實施例1、2係利用ASC-CM的MDCK及NIH-3T3細胞生長。 First, please refer to Figures 1 to 8 and the following detailed description: The present invention discloses a composition and method for treating cell growth using velvet antler stem cell-conditioned medium, and Examples 1 and 2 use MDCK and NIH-3T3 cells grown using ASC-CM.
從健康梅花鹿採集鹿茸,從其採集大約10毫米×5毫米×5毫米大小的小鹿茸尖端段;採集的鹿茸尖端段用75%酒精清洗一次,然後用磷酸鹽緩衝生理鹽水(PBS)清洗兩次;然後在10公分培養皿中將鹿茸尖端段切成1毫米的較小段,再次用PBS進行清洗;然後將小切片以速率200xG離心2分鐘,從中去除PBS並分離鹿茸幹細胞(ASC);將分離的ASC 以每孔1×105個細胞的密度植種在六孔板中;然後將1毫升的0.25%胰蛋白酶乙二胺四乙酸(EDTA)添加到ASC中並在37℃下培養1小時;接著添加補充有10%胎牛血清(FBS)的達爾伯克氏改良伊格爾氏培養基(Dulbecco's Modified Eagle Medium)(DMEM),將鹿茸幹細胞(ASC)培養72小時,其間每2至3天更換一次生長培養基;然後採集上清液作為鹿茸幹細胞條件培養基(ASC-CM)以用於實驗。 Antlers were collected from healthy sika deer, and small antler tip segments of about 10 mm × 5 mm × 5 mm were collected from them; the collected antler tip segments were washed once with 75% alcohol, and then washed twice with phosphate buffered saline (PBS); then the antler tip segments were cut into 1 mm smaller segments in a 10 cm culture dish, and washed again with PBS; then the small slices were centrifuged at a speed of 200xG for 2 minutes, PBS was removed and antler stem cells (ASCs) were isolated; the isolated ASCs were plated at 1×10 ASCs were seeded at a density of 5 cells/well in a six-well plate. Then, 1 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) was added to ASCs and cultured at 37°C for 1 hour. Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was then added and ASCs were cultured for 72 hours, during which the growth medium was replaced every 2 to 3 days. The supernatant was then collected as ASC-CM for experiments.
在圖2中描繪麥登-達比犬類腎臟上皮細胞(MDCK細胞)實驗組的製備;將含有10%FBS的1毫升ASC-CM與含有10%FBS的4毫升DMEM混合以形成含有10%FBS、20%ASC-CM及70%DMEM的5毫升生長培養基;進一步將此5毫升的生長培養基與15毫升不含FBS的DMEM混合以形成含有2.5% FBS、5% ACM及92.5% DMEM的20毫升生長培養基;另外為要將細胞與上清液分離,將200公克的MDCK細胞離心約10分鐘;然後藉由離心過程分離的細胞以3×105個細胞/毫升的濃度置於不含FBS的DMEM中;然後將1毫升這種MDCK細胞DMEM培養物放入每個孔中,並用1毫升的上述20毫升生長培養基進行稀釋以形成含有MDCK細胞的1.25% FBS+2.5%ASC-CM及96.25%DMEM溶液。 FIG2 depicts the preparation of the Madden-Darby canine renal epithelial cell (MDCK cell) experimental group; 1 ml of ASC-CM containing 10% FBS was mixed with 4 ml of DMEM containing 10% FBS to form 5 ml of growth medium containing 10% FBS, 20% ASC-CM and 70% DMEM; the 5 ml of growth medium was further mixed with 15 ml of DMEM without FBS to form 20 ml of growth medium containing 2.5% FBS, 5% ACM and 92.5% DMEM; in addition, to separate the cells from the supernatant, 200 g of MDCK cells were centrifuged for about 10 minutes; then the cells separated by centrifugation were centrifuged at 3×10 5 cells/ml in DMEM without FBS; then 1 ml of this MDCK cell DMEM culture was placed in each well and diluted with 1 ml of the above 20 ml growth medium to form a 1.25% FBS + 2.5% ASC-CM and 96.25% DMEM solution containing MDCK cells.
MDCK細胞對照組的製備涉及含有2.5% FBS及97.5% DMEM的生長培養基的製備;接著將1毫升這種生長培養基在DMEM中與1毫升MDCK細胞混合以形成1.25%FBS及98.75%DMEM含有MDCK細胞的生長培養基。 Preparation of the MDCK cell control group involved the preparation of a growth medium containing 2.5% FBS and 97.5% DMEM; 1 ml of this growth medium was then mixed with 1 ml of MDCK cells in DMEM to form a growth medium containing 1.25% FBS and 98.75% DMEM containing MDCK cells.
將所有孔全在Forma系列II 3 110調溫培養箱中以37℃ 5x CO2在37℃下進行培養;在第1、3及5天從孔中得到樣品;將MDCK細胞用PBS清洗兩次,加入0.5毫升的胰蛋白酶以分離MDCK細胞,以便進行MDCK細胞計數;在圖3中顯示作為結果的計數,圖3中0%曲線表示不存在ASC-CM的對照組,而20%曲線表示ASC-CM包含20%培養基的實驗組。 All wells were cultured in a Forma Series II 3 110 temperature-controlled incubator at 37°C 5x CO2 ; samples were obtained from the wells on days 1, 3 and 5; the MDCK cells were washed twice with PBS, and 0.5 ml of trypsin was added to detach the MDCK cells for MDCK cell counting; the resulting counts are shown in Figure 3, where the 0% curve represents the control group without ASC-CM, and the 20% curve represents the experimental group in which ASC-CM contained 20% of the culture medium.
如圖3所示在培養5天後,在0%ASC-CM與20%ASC-CM培養基之間MDCK細胞計數中的差異大於20%;圖4顯示在顯微鏡下顯示0%ASC-CM培養基中細胞凋亡與20%ASC-CM培養基中MDCK細胞增殖的對照;圖3與圖4皆支持ASC-CM刺激MDCK細胞增殖的結果。 As shown in Figure 3, after 5 days of culture, the difference in MDCK cell counts between 0% ASC-CM and 20% ASC-CM culture media was greater than 20%; Figure 4 shows the comparison of cell apoptosis in 0% ASC-CM culture media and MDCK cell proliferation in 20% ASC-CM culture media under a microscope; both Figures 3 and 4 support the results that ASC-CM stimulates MDCK cell proliferation.
對NIH-3T3細胞進行相同的實驗,結果顯示在圖5及圖6中;如圖5所示即使在培養2天後,在0% ASC-CM與20% ASC-CM培養基之間MDCK細胞計數中的差異大於10%;第3天的細胞計數中的差異增加到20%以上,及第6天增加到近30%;圖6顯示在顯微鏡下顯示0%ASC-CM培養基中細胞凋亡與20% ASC-CM培養基中NIH-3T3細胞增殖的對照:圖5及圖6 皆支持ASC-CM刺激NIH-3T3細胞增殖的結果;可在人類視網膜色素上皮細胞(ARPE-19)及大鼠心肌原細胞(H9c2)上進行相同的實驗以證明在不同類型細胞的細胞生長上的治療作用。 The same experiment was performed on NIH-3T3 cells, and the results are shown in Figures 5 and 6. As shown in Figure 5, even after 2 days of culture, the difference in MDCK cell counts between 0% ASC-CM and 20% ASC-CM medium was greater than 10%. The difference in cell counts increased to more than 20% on day 3, and nearly 30% on day 6. Figure 6 shows the difference in cell apoptosis in 0% ASC-CM medium and 20% ASC-CM medium under a microscope. Comparison of NIH-3T3 cell proliferation in ASC-CM medium: Figures 5 and 6 Both support the results that ASC-CM stimulates NIH-3T3 cell proliferation; the same experiment can be performed on human retinal pigment epithelial cells (ARPE-19) and rat cardiomyocytes (H9c2) to demonstrate the therapeutic effect on cell growth of different cell types.
實施例3:利用PLGA顆粒遞送系統製備ASC-CM奈米顆粒: Example 3: Preparation of ASC-CM nanoparticles using PLGA particle delivery system:
在圖7中描繪用PLGA顆粒遞送系統製備ASC-CM奈米顆粒;此過程從製備含有ASC-CM的水相溶液及含有脂質的油相溶液開始;具體地水相溶液包含約200微升(μL)蛋白質溶液,該蛋白質溶液含有ASC-CM,以0.5%聚乙烯醇(PVA)磷酸鹽緩衝生理鹽水(PBS)溶液中約佔14.4毫克/毫升的濃度;油相溶液在2毫升的二氯甲烷(DCM)中含有200毫克的Resomer®RG502H聚(D,L-丙交酯-共-乙交酯)(RG502H PLGA),其中RG502H PLGA約佔油相溶液重量體積的10%。 FIG7 depicts the preparation of ASC-CM nanoparticles using a PLGA particle delivery system; the process begins with the preparation of an aqueous solution containing ASC-CM and an oily solution containing lipids; specifically, the aqueous solution comprises approximately 200 μL of a protein solution containing ASC-CM at a concentration of approximately 14.4 mg/mL in a 0.5% polyvinyl alcohol (PVA) phosphate buffered saline (PBS) solution; the oily solution contains 200 mg of Resomer® RG502H poly(D,L-lactide-co-glycolide) (RG502H PLGA) in 2 mL of dichloromethane (DCM), wherein RG502H PLGA accounts for approximately 10% of the weight volume of the oily solution.
接著使水相溶液與油相溶液在均質機(IKA)中混合,以約5的速度攪勻約1.5分鐘以形成油包水乳劑。隨後將該乳劑添加到約15毫升重量體積約5%的PVA溶液中,將作為結果的乳液再次以約5的速度攪勻約1.5分鐘以形成油包水雙乳劑。 The aqueous phase solution was then mixed with the oil phase solution in a homogenizer (IKA) and stirred at a speed of about 5 for about 1.5 minutes to form an oil-in-water emulsion. The emulsion was then added to about 15 ml of a 5% by weight PVA solution, and the resulting emulsion was again stirred at a speed of about 5 for about 1.5 minutes to form an oil-in-water double emulsion.
在下一步驟中接著將油包水雙乳劑以4℃進行溶劑 DCM蒸發過程約2小時以形成懸浮液;然後將懸浮液以速率約300xG離心約5分鐘以去除大的硬結塊;接著將約15.5毫升等分的上清液以速率約21,000xG離心約15分鐘;然後將奈米顆粒細圓粒清洗1次並以速率約500xG離心約3分鐘;將約15.5毫升等分的上清液再次以速率約21,000xG離心約15分鐘;然後使結果再懸浮在約1.9毫升無顆粒去離子水中;將懸浮液以約-80℃冷凍乾燥並儲存;或者將在約1.9毫升無顆粒去離子水中的ASC-CM奈米顆粒與約10毫升的25%硫酸鋁鈉及75%蔗糖混合,然後冷凍乾燥成粉末形式並封裝成可口服給藥的膠囊。 In the next step, the oil-in-water double emulsion was subjected to a solvent evaporation process at 4°C for about 2 hours to form a suspension; the suspension was then centrifuged at a rate of about 300xG for about 5 minutes to remove large hard lumps; then about 15.5 ml of the supernatant was centrifuged at a rate of about 21,000xG for about 15 minutes; then the nanoparticle pellets were washed once and centrifuged at a rate of about 500xG for about 3 minutes; about 15.5 ml of The aliquot of the supernatant is centrifuged again at a rate of about 21,000xG for about 15 minutes; the result is then resuspended in about 1.9 ml of particle-free deionized water; the suspension is freeze-dried at about -80°C and stored; or the ASC-CM nanoparticles in about 1.9 ml of particle-free deionized water are mixed with about 10 ml of 25% sodium aluminum sulfate and 75% sucrose, then freeze-dried into a powder form and packaged into capsules for oral administration.
然後將冷凍乾燥的奈米顆粒溶解在pH7.2的PBS緩衝液中,用以使用動態光散射(DLS)顯示特徵;在圖8中概述結果顯示Z平均直徑約為444.9奈米,PDT(光動力治療)則為0.265。 The freeze-dried nanoparticles were then dissolved in PBS buffer at pH 7.2 for characterization using dynamic light scattering (DLS); the results summarized in Figure 8 show a Z-average diameter of approximately 444.9 nm and a PDT (photodynamic therapy) of 0.265.
綜上所陳,本發明確實符合專利法第21條之成立要件,懇請 鈞局明鑒,惠予授准合法之專利權成立,至感德便。 In summary, this invention does meet the requirements of Article 21 of the Patent Law. We sincerely request the Bureau to review and grant the legal patent right. We are grateful.
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