方法

設備和(he)(he)材料。設備。使用(yong)96孔熒(ying)光板讀數器（Sepctra Max-Gemini EM，Molecular Devices，Sunnyvale，CA）和(he)(he)96淺孔熒(ying)光板（Greiner Bio-one，Kremsmuenster，Austria）。

材(cai)料。用于驗證該方法的所有(you)標準化(hua)(hua)合物(wu)均購自(zi)Fisher Scientific/AG Scientific/Aldrich/Acros/Tocris/MP Biomedicals或從Novartis存儲庫獲得，無需(xu)進一步(bu)純化(hua)(hua)即可使(shi)用。

緩沖器。pH 7.2 N-2-羥乙基哌嗪(qin)-N0-2-乙磺酸(suan)(HEPES)緩沖液、20 mM含(han)0.1 mM乙二胺四(si)乙酸(suan)(EDTA)（MP biomedicals,Solon,OH）和pH 4.8乙酸(suan)緩沖液，使用40 mM。

熒(ying)光(guang)染料。NN-Dimethyl-6-propionyl-2-naphthylamine(Prodan)購自Fisher Scientific(Fair Lawn,NJ)。

陰離子脂質。1,2-Dioctanoyl-sn-glycero-3-phospho-L-serine(sodium salt)(diC8PS)購自Avanti Polar Lipids Inc.(Alabaster,AL)。

樣(yang)品制備。測試化合(he)(he)物(wu)的(de)(de)儲備溶液。將測試化合(he)(he)物(wu)以10、1或0.1 mM的(de)(de)濃度(du)溶解在二甲基(ji)亞砜(feng)(DMSO)中。對于在上(shang)述濃度(du)下不能(neng)完全溶于DMSO的(de)(de)化合(he)(he)物(wu)如慶大霉素(su)，則使用蒸餾水代(dai)替(ti)。

脂(zhi)質溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)。從Avanti購買的(de)(de)(de)diC8PS氯仿中(zhong)的(de)(de)(de)50 mg/mL儲備溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)在(zai)(zai)(zai)氯仿/甲(jia)醇(5:1 v/v)中(zhong)進一步(bu)稀釋(shi)(shi)，得(de)到(dao)16 mg/mL的(de)(de)(de)濃度(du)，不使(shi)用(yong)(yong)時儲存(cun)在(zai)(zai)(zai)80°C。將(jiang)4 mL該(gai)溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)轉(zhuan)移到(dao)一個干(gan)凈的(de)(de)(de)20 mL玻璃小瓶(ping)中(zhong)，并(bing)在(zai)(zai)(zai)溫和(he)(he)的(de)(de)(de)氮氣流下蒸(zheng)發溶(rong)(rong)(rong)(rong)(rong)(rong)劑。將(jiang)干(gan)燥的(de)(de)(de)脂(zhi)質膜分散在(zai)(zai)(zai)10 mL的(de)(de)(de)20 mM HEPES緩(huan)沖(chong)液(ye)(ye)(ye)(ye)(ye)、0.1 mM EDTA、pH 7.2中(zhong)，最終濃度(du)為6 mg/mL。使(shi)用(yong)(yong)前(qian)將(jiang)溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)劇烈渦旋，置于60℃水(shui)浴中(zhong)孵育45分鐘。該(gai)溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)在(zai)(zai)(zai)不使(shi)用(yong)(yong)時儲存(cun)在(zai)(zai)(zai)4°C，然后在(zai)(zai)(zai)使(shi)用(yong)(yong)前(qian)用(yong)(yong)上述(shu)相同的(de)(de)(de)HEPES緩(huan)沖(chong)液(ye)(ye)(ye)(ye)(ye)稀釋(shi)(shi)以(yi)獲得(de)4和(he)(he)1 mg/mL溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)。染料溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)。Prodan溶(rong)(rong)(rong)(rong)(rong)(rong)解在(zai)(zai)(zai)甲(jia)醇中(zhong)制(zhi)成0.3 mg/mL的(de)(de)(de)儲備溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)。溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)的(de)(de)(de)容器用(yong)(yong)箔包裹(guo)并(bing)在(zai)(zai)(zai)室(shi)溫下避(bi)光儲存(cun)。在(zai)(zai)(zai)加入樣品溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)之前(qian)，使(shi)用(yong)(yong)甲(jia)醇將(jiang)溶(rong)(rong)(rong)(rong)(rong)(rong)液(ye)(ye)(ye)(ye)(ye)稀釋(shi)(shi)至(zhi)0.05 mg/mL。

圖2.在不同(tong)濃(nong)度(du)的脂(zhi)質diC8PS下記錄的熒光發射，范圍從64μg/mL到3.47 mg/mL。在對(dui)照(zhao)中，添加純DMSO代替測試(shi)化合物(O)，1.1 mg/mL的急劇上升標志著diC8PS的CMC。將10μL的10 mM硫利達嗪(qin)(9)引入基質（20 mM HEPES緩(huan)沖(chong)液中的diC8PS），CMC轉移(yi)到較低濃(nong)度(du)（0.042 mg/mL）。RFU：相對(dui)熒光單位(wei)。

樣品板(ban)制(zhi)備(bei)(bei)(bei)和測量(liang)。通(tong)(tong)過(guo)混合(he)10μL化合(he)物儲(chu)備(bei)(bei)(bei)溶(rong)液(ye)(ye)（用于(yu)對(dui)(dui)照的(de)(de)純(chun)DMSO）、10μL Prodan溶(rong)液(ye)(ye)和正確(que)量(liang)的(de)(de)脂質(zhi)溶(rong)液(ye)(ye)制(zhi)備(bei)(bei)(bei)樣品板(ban)，使最(zui)終(zhong)脂質(zhi)濃(nong)(nong)度(du)為(wei)3.3、2、1、0.8、0.5、0.25、0.1、0.08、0.06、0.006和0.0006 mg/mL，每孔總體積為(wei)110μL，通(tong)(tong)過(guo)添加緩沖溶(rong)液(ye)(ye)進行調整。來自10、1和0.1 mM DMSO儲(chu)備(bei)(bei)(bei)溶(rong)液(ye)(ye)的(de)(de)最(zui)終(zhong)藥物濃(nong)(nong)度(du)分別為(wei)910、91和9.1μM。使用Spectra Max-Gemini EM讀(du)板(ban)器讀(du)取板(ban)，激發(fa)(fa)波長(chang)為(wei)360 nm，發(fa)(fa)射(she)波長(chang)為(wei)430 nm。臨界膠束濃(nong)(nong)度(du)的(de)(de)測定(ding)。將熒光讀(du)數(shu)與脂質(zhi)濃(nong)(nong)度(du)作圖。CMC被定(ding)義(yi)為(wei)濃(nong)(nong)度(du)點，高于(yu)該濃(nong)(nong)度(du)點熒光信號的(de)(de)增強(qiang)是濃(nong)(nong)度(du)的(de)(de)函數(shu)，如圖1所示。雙相(xiang)線性圖的(de)(de)截距(ju)由諾華IT開發(fa)(fa)的(de)(de)內部Microsoft Excel工作表(biao)自動確(que)定(ding)。Microsoft Excel工作表(biao)通(tong)(tong)過(guo)從數(shu)據中的(de)(de)點的(de)(de)移(yi)動窗口中尋找最(zui)佳R2值來定(ding)位(wei)最(zui)適(shi)合(he)垂直(zhi)線性曲(qu)線的(de)(de)值。其余數(shu)據用于(yu)水(shui)平線性曲(qu)線。通(tong)(tong)過(guo)從垂直(zhi)曲(qu)線中減去(qu)水(shui)平曲(qu)線來計(ji)算截距(ju)點，即CMC值。對(dui)(dui)照實(shi)驗中的(de)(de)交(jiao)點代表(biao)培養基基質(zhi)中脂質(zhi)的(de)(de)CMC。

測(ce)(ce)定(ding)(ding)重(zhong)現性。為了(le)測(ce)(ce)試(shi)測(ce)(ce)定(ding)(ding)的(de)再現性，一些化合(he)物在不同的(de)測(ce)(ce)定(ding)(ding)日(ri)期進行(xing)了(le)多(duo)次(ci)測(ce)(ce)試(shi)。連同在每個測(ce)(ce)定(ding)(ding)日(ri)期運行(xing)的(de)對(dui)照(zhao)數據，變異(yi)系(xi)數(CV)的(de)范圍(wei)為4%到8%，表明當前方法具有(you)良好的(de)重(zhong)現性（圖1）。

Delta-8使用新方法測試CMC，而不是表面張力測試法——摘要

Delta-8使用新方法測試CMC，而不是表面張力測試法——方法

Delta-8使用新方法測試CMC，而不是表面張力測試法——結果

Delta-8使用新方法測試CMC，而不是表面張力測試法——討論