TWI572369B - 酸鹼應答型奈米微粒的製備並應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途 - Google Patents

酸鹼應答型奈米微粒的製備並應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途 Download PDF

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TWI572369B
TWI572369B TW104119949A TW104119949A TWI572369B TW I572369 B TWI572369 B TW I572369B TW 104119949 A TW104119949 A TW 104119949A TW 104119949 A TW104119949 A TW 104119949A TW I572369 B TWI572369 B TW I572369B
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acid
dox
histidine
base responsive
loaded
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TW201700093A (zh
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邱信程
姜文軒
洪嘉謙
余亭葦
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國立清華大學
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Description

酸鹼應答型奈米微粒的製備並應用於製備促進抗癌藥物於腫瘤的傳輸 與深層滲透之藥物的用途
本發明涉及抗腫瘤藥物，尤指一種可應用於強效滲透腫瘤組織之酸鹼應答型奈米微粒。
目前任何一種治療癌症的藥物都有明顯的副作用例如頭暈、掉髮、皮膚老化等等症狀，因此，研究人員提出了標靶治療的概念，具體地說係利用標靶藥物並經由體內血液循環累積於癌症腫瘤組織，以達到抑制腫瘤細胞增長的功效，目前這類所謂藥物控制釋放的技術已大量的被醫學界所重視，而市面上的標靶藥物仍存在著不少待改進的缺點，於是該如何降低副作用、增加藥物包覆量、將藥物準確的釋放在腫瘤位置仍是研究人員努力欲解決的問題。
近年來高分子微胞被廣泛應用於醫藥界領域，高分子微胞是由兩性高分子鏈段所組成，一端帶有親水性鏈段，另一端則為親油性鏈段，兩性高分子鏈段中的親油性鏈段在水相中會透過凡德瓦力結合成一核心疏水區，該區域可作為脂溶性藥物的儲存槽，親水性鏈段則位於疏水核心的外部以增進高分子微胞於水相中的結構穩定性。然而，已發展的高分子微胞在高度稀釋的血液循環系統中仍存在結構安定性不佳與較差的腫瘤累積的能力。因此，如何設計出結構穩定性佳且能有效累積於腫瘤病灶並傳遞抗腫瘤 藥物的奈米載體仍是研究人員欲解決的問題。
為解決目前市面上的標靶藥物所面臨藥物包覆量較低以及無法準確將抗癌藥物大量累積於腫瘤區域的難題，本發明提供了一種酸鹼應答型奈米微粒，可經由Enhanced permeability and retention(EPR)effect累積至腫瘤微酸環境後，透過其表面電性的轉換(由負電性趨向電中性)來增強酸鹼應答型奈米微粒與癌細胞間的交互作用力並促進其滲透至腫瘤深層區域的能力，進而增加酸鹼應答型奈米微粒在腫瘤區域的累積量與被癌細胞攝取程度，此舉可將抗癌藥物大量傳輸至癌細胞中以提高癌症治療效果。
為達上述目的，本發明採用以下技術方案：一種酸鹼應答型奈米微粒，為一酸鹼應答型高分子材料與聚乳酸聚甘醇酸於水相中經自組裝形成，其特徵在於，該酸鹼應答型高分子材料係由一聚乙二醇衍生物與一組胺酸衍生物經化學反應所形成，其中該酸鹼應答型奈米微粒進一步包含親水區以及疏水區。
其中該聚乙二醇衍生物與該組胺酸衍生物經由酯化反應所合成該酸鹼應答型高分子材料。
其中該聚乙二醇衍生物為維他命E(TPGS)或二硬脂基磷脂乙醇胺(DSPE-PEG)。
其中該組胺酸衍生物為N-乙醯基組胺酸(N-acetyl-Histidine)、L-組胺酸(L-Histidine)、D-組胺酸(D-Histidine)或3-甲基組胺酸(3-Methyl-L-histidine)。
其中該疏水區進一步包含抗癌藥物、顯影劑、光熱藥劑、奈米金屬粒子或其組合。
為使能更進一步瞭解本發明之特徵及技術內容，請參閱以下有關本發明之詳細說明與附圖，但是此等說明與所附圖式僅係用來說明本發明，而非對本發明的權利範圍作任何的限制。
S401‧‧‧將DOX-HCL溶於DMSO避光攪拌
S402‧‧‧將NAcHis-TPGS溶於DMSO後，滴入磷酸鹽緩衝溶液
S403‧‧‧將DOX與PLGA滴入S402所得產物
S404‧‧‧利用透析移除未包覆DOX
S901‧‧‧將DOX-HCL溶於DMSO避光攪拌
S902‧‧‧將NAcHis-TPGS溶於DMSO後，滴入磷酸鹽緩衝溶液
S903‧‧‧將DOX、ICG與PLGA滴入S902所得產物
S904‧‧‧利用透析移除未包覆DOX、ICG
30、40‧‧‧藥物傳輸系統
31、41‧‧‧正常細胞
32、42‧‧‧腫瘤細胞
33‧‧‧腫瘤細胞之細胞核
501‧‧‧實驗組NHTPNs
502‧‧‧實驗組DOX-loaded NHTPNs
503‧‧‧實驗組DOX-loaded TPNs
701‧‧‧實驗組Free DOX
702‧‧‧實驗組DOX-loaded NHTPNs
703‧‧‧實驗組DOX-loaded TPNs
1101‧‧‧實驗組ICG/DOX-loaded NHTPNs
1102‧‧‧實驗組ICG/DOX-loaded TPNs
1103‧‧‧實驗組DOX-loaded NHTPNs
1104‧‧‧實驗組ICG-loaded NHTPNs
1105‧‧‧實驗組NHTPNs
1301‧‧‧實驗組ICG/DOX-loaded NHTPNs在pH 7.4
1302‧‧‧實驗組ICG/DOX-loaded NHTPNs在pH 6.3
1303‧‧‧實驗組ICG/DOX-loaded TPNs在pH 7.4
1304‧‧‧實驗組ICG/DOX-loaded TPNs在pH 6.3
1401‧‧‧實驗組Free ICG
1402‧‧‧實驗組ICG-loaded NHTPNs
1403‧‧‧實驗組ICG/DOX-loaded TPNs
1404‧‧‧實驗組ICG/DOX-loaded NHTPNs
1501‧‧‧實驗組ICG/DOX-loaded NHTPNs
1502‧‧‧實驗組ICG/DOX-loaded TPNs
1503‧‧‧實驗組ICG-loaded NHTPNs
1504‧‧‧實驗組Free ICG
1505‧‧‧實驗組Free DOX
1506‧‧‧實驗組PBS
1601‧‧‧實驗組ICG/DOX-loaded NHTPNs+laser
1602‧‧‧實驗組ICG/DOX-loaded NHTPNs
1603‧‧‧實驗組ICG/DOX-loaded TPNs+laser
1604‧‧‧實驗組ICG-loaded NHTPNs+laser
1605‧‧‧實驗組Free ICG+laser
1606‧‧‧實驗組Free DOX+laser
1607‧‧‧實驗組PBS+laser
圖1是本發明實施例的NAcHis-TPGS合成示意圖。
圖2是本發明實施例的NAcHis-TPGS之H-NMR光譜圖。
圖3是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)之製備示意圖。
圖4是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)之製備步驟圖。
圖5是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)與對照組之界面電位圖。
圖6是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)之藥物傳輸系統。
圖7是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)與對照組之HeLa cell細胞內DOX濃度圖。
圖8是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之製備示意圖。
圖9是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之製備步驟圖。
圖10是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之粒徑分布圖。
圖11是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)與對照組之界面電位圖。
圖12是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之藥物傳輸系統。
圖13是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)於TRAMP-C1細胞內偵測DOX之流式細胞儀分析圖。
圖14是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之ICG累積於小鼠身體內部各器官之比例圖。
圖15是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之時間-溫度圖。
圖16是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之時間-腫瘤體積圖。
本發明將有利於酸鹼應答型奈米微粒經由Enhanced permeability and retention(EPR)effect累積至腫瘤微酸環境後，透過其表面電性由負電轉為電中性或正電之行為來增強酸鹼應答型奈米微粒與癌細胞(細胞膜為負電)間的交互作用力，進而自發性的增強在腫瘤區域的累積量與滲透腫瘤的能力以促進抗癌藥物被癌細胞吞噬的效率及癌症治療效果，本發明無須採用複雜製程與過多有機溶劑，且電位轉換過程並無牽涉分子脫離與水解，故沒有副產物的生成。
在下文中，將藉由圖式說明本發明之各種實施例來詳細描述本發明的酸鹼應答型奈米微粒。然而，本發明概念可能以許多不同形式來體現，且不應解釋為限於本文中所闡述之例示性實施例。此外，在圖式中相同參考數字可用以表示類似的製程步驟。
本發明提供一種酸鹼應答型奈米微粒，其中酸鹼應答型高分子材料係由聚乙二醇衍生物與組胺酸衍生物(R-Histidine)所組成，該聚乙二醇衍生物為聚乙二醇琥珀酸酯(D-α-tocopheryl polyethylene glycol 1000 succinate，TPGS)或二硬脂基磷脂乙醇胺-聚乙二醇(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)，DSPE-PEG)，其中該組胺酸衍生物(R-Histidine)為N-乙醯基組胺酸(N-acetyl-Histidine)、L-組胺酸(L-Histidine)、D-組胺酸(D-Histidine)、3-甲基組胺酸(3-Methyl-L-histidine)。
【第一實施例】
請參閱圖1以及圖2所示，本發明之一實施例所使用之酸鹼 應答型高分子為利用N-乙醯基組胺酸(N-Acetyl Histidine)修飾上聚乙二醇琥珀酸酯(D-α-tocopheryl polyethylene glycol 1000 succinate，TPGS)，合成物簡稱NAcHis-TPGS；本實施例中，合成方法是利用Steglich酯化反應，具體步驟如下：將TPGS、N-乙醯基-L-組氨酸(N-acetyl-1-histidine)、二環己基碳二亞胺(dicyclohexylcarbodiimide，DCC)、以及少量的4-二甲氨基吡啶(4-Dimethylaminopyridine，DMAP)溶於10mL之無水二甲基亞碸(Dimethyl sulfoxide，簡稱DMSO)中，並於40℃下攪拌48小時。而理想化條件為，N-acetyl-1-histidine之添加量約為TPGS莫耳數的5倍，且DCC之添加量約為TPGS莫耳數的8倍。
移除上述實驗過程中產生的二環已基尿素(Dicyclohexylurea，DCU)與DMSO。本實施例主要是利用抽氣過濾將DCU移除，並將產物(金黃色液體)置入透析袋(MWCO 1000)內，於室溫下對DMSO透析三天，用以移除溶液內未成功進行合成反應之殘留反應物，再對去離子水透析三天，移除溶液內的DMSO。
將上述溶液冷凍乾燥後得到產物為N-乙醯基組胺酸(N-acetyl-1-histidine)修飾上聚乙二醇琥珀酸酯(TPGS)，合成物簡稱NAcHis-TPGS。
如圖2所表示NAcHis-TPGS之H-NMR光譜圖，將純化後的NAcHis-TPGS溶於DMSO-d6溶劑，利用500MHz的核磁共振光譜儀定量分析高分子的化學組成，TPGS末端的甲基基團(peak a)之氫質子特徵峯化學位移為0.8ppm，N-Acetyl-Histidine之imidazole基團C-4碳(peak m)上的氫質子化學位移則在7.5ppm，計算特徵峰a與m的積分面積比值，與理論單元上的總氫數進行比較，即可獲得N-Acetyl-Histidine鍵結於TPGS上的效率，經計算後，TPGS的末端基改質率約為94.7%。產率則為88.0%。
請同時參閱圖3以及圖4所示，圖3是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)之製備示意圖，圖4是本發 明實施例的酸鹼應答型奈米微粒(DOX-loaded NAcHis-TPGS/PLGA nanoparticles(NHTPNs))之製備步驟圖。
在步驟S401中，先將鹽酸阿黴素(DOX．HCl)溶於DMSO後再加入triethyl amine於室溫下避光攪拌48小時後備用，此舉目的為增加DOX的疏水性以提高藥物包覆率，本發明不以抗癌藥物種類為限。而理想化條件為，triethyl amine的添加量為DOX莫耳數之2倍。
在步驟S402中，將預先改質完成的NAcHis-TPGS溶於DMSO(0.05mL)之後滴入pH 7.4，0.01M磷酸鹽緩衝溶液(phosphate buffer，3.5mL)中劇烈攪拌5分鐘(轉速設定為1350rpm)。而理想化條件為，NAcHis-TPGS的添加量為PLGA之80wt%。
在步驟S403中，於攪拌中緩慢將預先溶於DMSO(0.45mL)中的PLGA(poly(lactic-co-glycolic acid))(LA/GA=85/15(mol比例)，2.0mg)及DOX滴入於步驟S402中所得產物，滴入後持續劇烈攪拌30分鐘。而理想化條件為，DOX的添加量為PLGA之20wt%。
在步驟S404中，將均勻混和後之溶液於室溫靜置平衡30分鐘後置入透析模(MWCO 12000~14000)中，於4℃下對pH 7.4，0.01M phosphate buffer透析一天以移除未包覆之DOX與溶劑DMSO，將透析後的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)溶液保存於4℃冰箱中待後續實驗使用。
由圖3之示意圖可知，在液相中經自組裝形成的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)為奈米級的粒子，其中該酸鹼應答雙性高分子NAcHis-TPGS的PEG親水端位於載藥疏水核心(由裝載DOX之PLGA所構成)的外部。
為了對照比較，依照相同程序分別製備DOX-loaded TPGS/PLGA Nanoparticles(DOX-loaded TPNs)與未載藥的奈米粒子(NHTPNs)，其用以作為實驗對照組。
圖5是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)與對照組之界面電位圖。界面電位圖分析結果顯示實驗組DOX-loaded NHTPNs 502以及實驗組NHTPNs 501表面之histidine基團於弱酸水相中之大幅質子化確能促使微粒表面電性由負電趨向正電，相較之下，實驗組DOX-loaded TPNs 503則無電位轉換的特性。另外，在酸鹼值由pH 6.6下降至pH 6.0時，於電位變換過程中，實驗組DOX-loaded NHTPNs 502傾向產生粒子間的聚集，此一特性將有利於增進本發明之酸鹼應答型奈米微粒(DOX-loaded NHTPNs)在活體腫瘤的累積程度，並將抗癌藥物釋放出來進而毒殺癌細胞。
圖6是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)之藥物傳輸系統30，由圖中可示，其酸鹼應答型奈米微粒(DOX-loaded NHTPNs)並不會滲透在正常細胞31中，相對的，結合於PLGA核心外部的NAcHis-TPGS於腫瘤細胞32酸性環境中可藉由histidine基團上之imidazole官能基大幅質子化增加微粒表面的正電荷，此舉將有利於酸鹼應答型奈米微粒(DOX-loaded NHTPNs)經由EPR effect累積至腫瘤細胞32微酸環境時，透過其酸鹼應答型奈米微粒(DOX-loaded NHTPNs)表面電性由負電轉為正電的特性增進被腫瘤細胞32吞噬的效率，再將抗癌藥物DOX釋放至腫瘤細胞之細胞核33以提高毒殺癌細胞效果。
圖7是本發明實施例的酸鹼應答型奈米微粒(DOX-loaded NHTPNs)與對照組之HeLa cells內DOX濃度圖。將HeLa cells與酸鹼應答型奈米微粒(DOX-loaded NHTPNs)分別於微酸環境(pH 6.5、pH 6.0)及正常培養條件下(pH 7.4)共培養2小時後，利用微量螢光盤式儀測定胞內藥物濃度，分析結果顯示實驗組DOX-loaded NHTPNs 702在弱酸狀態下能大幅提高被癌細胞的攝入量，因此顯著提升癌細胞內DOX濃度。
在正常培養條件下(pH 7.4)相比，本發明的酸鹼應答型奈米微 粒(DOX-loaded NHTPNs)在微酸環境(pH 6.0)胞內藥物累積量可提升至少4倍以上，實驗組DOX-loaded TPNs 703在不同酸鹼值下HeLa cells內DOX濃度並無顯著變化，實驗組Free DOX 701在不同酸鹼值下HeLa cells內DOX濃度也同樣無顯著改變。上述實驗結果意味著將NAcHis-TPGS鑲嵌於酸鹼應答型奈米微粒(DOX-loaded NHTPNs)的表面確實能賦予其酸鹼應答表面電荷變換功能，進一步促進酸鹼應答型奈米微粒(DOX-loaded NHTPNs)在弱酸環境中被癌細胞的內吞效率。
【第二實施例】
請同時參閱圖8與圖9，圖8是本發明另一實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之製備示意圖，圖9是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之製備步驟圖。
在步驟S901中，將DOX．HCl溶於DMSO再加入triethyl amine於室溫避光下攪拌24小時後備用，此舉主要是增加DOX的疏水性以提高藥物包覆效率。而理想化條件為，triethyl amine的添加量為DOX莫耳數之2倍。
在步驟S902中，將含有NAcHis-TPGS的DMSO溶液，在磁石攪拌下緩慢滴入於磷酸緩衝溶液(pH 7.4,0.01M)，轉速設定為1350rpm攪拌五分鐘。而理想化條件為，將50μL DMSO溶液其中含有1.8mg NAcHis-TPGS(進料量為PLGA進料之80wt%)，在磁石攪拌下緩慢滴入於2.8mL磷酸緩衝溶液。
在步驟S903中，將含有PLGA(LA/GA：85/15)、DOX與光熱藥劑indocyanine green(ICG)的DMSO溶液，緩慢滴入含NAcHis-TPGS之磷酸緩衝溶液(DMSO/Water(v/v)=1/7)，於避光環境下磁石攪拌30分鐘。而理想化條件為，350μL DMSO溶液其中包含2mg PLGA、0.4mg DOX以及0.4mg ICG。
在步驟S904中，將步驟S903之產物裝入透析袋(MWCO 12000~14000)內對磷酸緩衝溶液透析24小時，以移除未包覆之藥物與DMSO，即可得酸鹼應答型奈米微粒ICG/DOX-loaded NAcHis-TPGS/PLGA Nanoparticles(簡稱ICG/DOX-loaded NHTPNs)水溶液並將透析後保存於4℃冰箱中待後續實驗使用。
為了對照比較，依照相同程序分別製備ICG/DOX-loaded TPGS/PLGA Nanoparticles(ICG/DOX-loaded TPNs)、ICG-loaded NHTPNs與未載藥之奈米粒子。
圖10是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之粒徑分布圖。隨著溶液pH值的降低，由pH 7.4降至pH 5，其粒徑並未因表面電性改變而變化，粒徑分佈圖顯示出酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)仍保持約50nm，突顯其良好的微粒物理穩定性。憑藉著本發明之酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)於弱酸水相中擁有較小的粒徑且表面趨於電中性的特點，能夠展顯出其長效於體內血液循環滯留時間，同時提高腫瘤組織的滲透性與被癌細胞吞噬的能力，以大幅提升癌細胞內的藥物濃度。
圖11是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)與對照組之界面電位圖，圖中各對照組為實驗組ICG/DOX-loaded NHTPNs 1101、實驗組ICG/DOX-loaded TPNs 1102、實驗組DOX-loaded NHTPNs 1103、實驗組ICG-loaded NHTPNs 1104以及實驗組NHTPNs 1105。
隨著溶液pH值的變化由7.4降至5.0，觀察實驗組ICG/DOX-loaded NHTPNs 1101、實驗組DOX-loaded NHTPNs 1103、實驗組ICG-loaded NHTPNs 1104與實驗組NHTPNs 1105的實驗結果後可明顯發現，表面具有NAcHis-TPGS之搭載雙藥之實驗組ICG/DOX-loaded NHTPNs 1101、搭載單藥之實驗組DOX-loaded NHTPNs 1103、搭載單藥之實驗組ICG-loaded NHTPNs 1104與未 搭載藥物之實驗組NHTPNs 1105的界面電位均由負電位趨於不帶電或正電位，說明微粒表面電性從負電轉變成電中性或帶有正電荷，此現象有助於其累積在微酸腫瘤區域並增進被癌細胞攝取的效率，實驗組ICG/DOX-loaded TPNs 1102因表面未具有NAcHis而不具酸鹼應答的特性。
此一界面電位轉變現象主要歸因於酸鹼應答型奈米微粒在中性環境(pH 7.4)，由於表面亦吸附水相中的負離子而帶些微負電，但是當周遭環境變為弱酸性時，水相中的氫離子將促使酸鹼應答型奈米微粒表面的histidine基團因imidazole官能基質子化而造成酸鹼應答型奈米微粒表面的正電荷大幅增加，進而賦予其具有富含正電荷的表面。
如此明顯的電荷轉換效果有利於酸鹼應答型奈米微粒於血液循環時(pH 7.4)，不會因吸附過多血液中的蛋白質而被免疫細胞清除，且有助於酸鹼應答型奈米微粒進入腫瘤微酸環境時，藉由其富含正電荷的表面與帶負電之細胞膜間交互作用力的增加而增進被癌細胞攝取的程度。此外，因為ICG分子具有兩個磺酸根結構(pKa約2.0)，故實驗組ICG-loaded NHTPNs 1104在pH 7.4至5.0範圍內均帶負電。當ICG與NAcHis-TPGS共同嵌入疏水PLGA核心表面時，將無可避免的衝擊搭載ICG奈米微粒的電荷轉換能力。雖然如此，實驗組ICG/DOX-loaded NHTPNs 1101與實驗組ICG-loaded NHTPNs 1104在微酸的環境下(pH 6.0)仍能保持較佳的電荷轉換能力，使其表面從負電荷轉變成趨於電中性，此舉同樣有助於累積於癌細胞。
圖12是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之藥物傳輸系統40。當酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)於腫瘤微環境中(pH 7.0~6.0)，可因其表面部分質子化(protonation)之histidine單元增加與腫瘤細胞42之靜電荷交互作用力，進而提升被腫瘤細胞42吞噬的程度。 由於酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)本身粒徑較小、趨近電中性的表面，不僅能提高被腫瘤細胞42內吞的程度，且也能增加被腫瘤相關巨噬細胞吞噬的機會，進一步抵達缺氧區進而毒殺腫瘤細胞42。
圖13是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)於TRAMP-C1細胞內偵測DOX之流式細胞儀分析圖。另外，由流式細胞儀(flow cytometry)的分析數據可發現TRAMP-C1細胞與實驗組ICG/DOX-loaded NHTPNs在pH 6.3 1302共培養於較酸的環境中其細胞的DOX螢光強度明顯高於其實驗組ICG/DOX-loaded NHTPNs在pH 7.4 1301的培養條件，以及明顯高於其奈米微面表面未鑲嵌NAcHis之實驗組ICG/DOX-loaded TPNs在pH 7.4 1303與實驗組ICG/DOX-loaded TPNs在pH 6.3 1304。因此，將NAcHis-TPGS鑲嵌於奈米微粒的表面確實能賦予其表面酸鹼應答電荷變換功能，進一步促進酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)在弱酸環境中被癌細胞的內吞效率。
請同時參照圖12和圖14，圖14是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之ICG累積於小鼠身體內部各器官之比例圖。經尾靜脈注射48小時後之活體觀察實驗結束後，將動物犧牲取其主要器官及腫瘤，利用非侵入性活體影像系統(IVIS)偵測各組織的ICG螢光表現。實驗組Free ICG 1401於腫瘤沒有螢光表現，其螢光大多出現在肝臟及腫瘤位置，其實驗結果符合多數文獻報導肝臟是ICG主要的代謝器官。
值得注意的是，酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)組別顯示其累積在腫瘤的螢光強度明顯高於肝臟，並且實驗組ICG/DOX-loaded NHTPNs 1404在腫瘤的螢光強度明顯高於實驗組Free ICG 1401、實驗組ICG-loaded NHTPNs 1402與實驗組ICG/DOX-loaded TPNs 1403。由上述可知，於腫瘤微酸環境中 具有表面電荷轉換能力的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)不僅能避免ICG迅速被肝臟代謝而且有效促進ICG於腫瘤部位的累積，提供良好腫瘤影像與後續光熱治療進而抑制腫瘤生長。
請參閱圖15，圖15是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之時間-溫度圖，除了針對實驗組ICG/DOX-loaded NHTPNs 1501、實驗組ICG-loaded NHTPNs 1503與實驗組ICG/DOX-loaded TPNs 1502進行隨時間溫度變化評估外，亦採用實驗組PBS 1506、實驗組Free ICG 1504、實驗組Free DOX 1505作為實驗的對照組。經近紅外光雷射(NIR laser)照射腫瘤，因實驗組PBS 1506與實驗組Free DOX 1505缺乏光熱轉換特性故沒有展現出明顯腫瘤升溫的情況。
值得一提的是，實驗組ICG/DOX-loaded NHTPNs 1501與實驗組ICG-loaded NHTPNs 1503的組別於腫瘤部位則有顯著的升溫效果，最高溫度可分別達到60.2℃與56.3℃。另外，實驗組ICG/DOX-loaded TPNs 1502的組別於腫瘤最高溫度則為50.3℃。上述結果也呼應非侵入性活體影像系統(IVIS)所測得之利用NHTPNs載體來傳輸ICG確能更有效將ICG累積至腫瘤部位，進而展現出優異的光熱局部升溫效應來抑制腫瘤生長。從圖15明顯得知，實驗組Free ICG 1504在相同條件下，因其無法有效藉由EPR effect累積至腫瘤部位，且實驗組Free ICG 1504在血液循環過程中易被快速清除，故光熱升溫效果不佳，最高溫度僅達到41.4℃。
圖16是本發明實施例的酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)之時間-腫瘤體積圖。在施予紅外光雷射光照射後，為了觀察腫瘤抑制效果，本實驗進行腫瘤體積與小鼠體重的監測直到給藥後15天。將各組別腫瘤體積與各組別最初(day 0)的腫瘤體積標準化，得到腫瘤相對體積隨時間變化趨勢圖。 結果顯示實驗組PBS+laser 1607、實驗組Free ICG+laser 1605與實驗組Free DOX+laser 1606經紅外光雷射照射後，並沒有明顯的腫瘤生長抑制效果，源自於未經包覆的DOX與ICG無法有效的累積於腫瘤部位，故無法透過化療或是光觸發產熱抑制腫瘤生長，在15天後小鼠之腫瘤相對體積增大約22~26倍。
相比之下，經紅外光雷射照射之實驗組ICG/DOX-loaded NHTPNs+laser 1601與ICG-loaded NHTPNs+laser 1604組別在1~7天的療程中表現出相似腫瘤生長抑制效果。值得注意的是在第8~15天的療程中，相較於實驗組ICG/DOX-loaded NHTPNs+laser 1601的組別，實驗組ICG-loaded NHTPNs+laser 1604的小鼠其腫瘤復發程度較為顯著。
上述研究結果說明實驗組ICG-loaded NHTPNs+laser 1604有效的累積於腫瘤部位並經紅外光雷射照射後，所產生的高熱在治療初期能對腫瘤癌細胞造成致死熱傷害，但由於僅單次給藥與光照所產生的光熱治療無法全面殺死癌細胞而造成後續的腫瘤復發。
值得一提的是，對於施予實驗組ICG/DOX-loaded NHTPNs+laser 1601的組別來說，初期的強力光熱治療搭配後續的化療，即PLGA的持續降解造成酸鹼應答型奈米微粒(ICG/DOX-loaded NHTPNs)的核心結構較為鬆散，爾後促進DOX釋放，能更有效的抑制癌細胞增生而達到較佳的腫瘤生長抑制效果。此外，本實驗也發現若實驗組ICG/DOX-loaded NHTPNs 1602沒有施予紅外光雷射照射，意即僅依靠DOX之化學治療，其腫瘤抑制效果遠遜於施加紅外光雷射照射之實驗組ICG/DOX-loaded NHTPNs+laser 1601。
【第三實施例】
本發明另一實施例的酸鹼應答型奈米微粒為首先將DOX． HCl溶於DMSO再加入triethyl amine於室溫避光下攪拌24小時後備用，此舉主要是增加DOX的疏水性以提高藥物包覆效率。而理想化的條件為，triethyl amine的添加量為DOX莫耳數之4倍。
將含有1.8mg NAcHis-TPGS的DMSO溶液，在磁石攪拌下滴入分散於磷酸緩衝溶液(pH 7.4,0.01M)，轉速設定為1350rpm攪拌五分鐘。而理想化的條件為，50μL的DMSO溶液其中包含1.8mg NAcHis-TPGS(進料量為PLGA進料之80wt%)滴入2.8mL的磷酸緩衝溶液。
接著，再將含有PLGA(LA/GA：85/15)、DOX與奈米金屬粒子SPION(superparamagnetic iron oxide Nanoparticles，SPION)的DMSO溶液，緩慢滴入含NAcHis-TPGS之磷酸緩衝溶液(DMSO/Water(v/v)=1/7)，於避光環境下磁石攪拌30分鐘。而理想化的條件為，350μL的DMSO溶液其中包含2mg PLGA、0.4mg DOX與0.2mg SPION。此外，奈米金屬粒子為顯影劑的其中一種類型，本發明不以奈米金屬粒子的種類為限。
之後將其溶液裝入透析袋(MWCO 12000~14000)內對磷酸緩衝溶液(pH 7.4,0.01M)透析24小時，以移除未包覆之藥物與DMSO，即可得酸鹼應答型奈米微粒(SPION/DOX-loaded NHTPNs)之水溶液並將透析後的酸鹼應答型奈米微粒溶液保存於4℃冰箱中。
【第四實施例】
將N-乙醯基組胺酸(N-acetyl Histidine)修飾上二硬脂基磷脂乙醇胺-聚乙二醇(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)，DSPE-PEG)(本實施例簡稱NAcHis-DSPE-PEG)，其的合成方法是利用Steglich酯化反應。
將DSPE-PEG、N-acetyl-1-histidine、二環己基碳二亞胺 (dicyclohexylcarbodiimide，DCC)、少量的4-二甲氨基吡啶(4-Dimethylaminopyridine，DMAP)溶於無水10mL二甲基亞碸(Dimethyl sulfoxide，簡稱DMSO)，於40℃攪拌48小時。而理想化的條件為，N-acetyl-1-histidine的添加量為TPGS莫耳數之5倍，DCC的添加量為TPGS莫耳數之8倍。
隨後利用抽氣過濾將DCU移除，並將產物置入透析袋(MWCO 1000)內，於室溫下對DMSO透析三天，用以移除溶液內殘留的反應物，再對去離子水透析三天，移除溶液內的DMSO，最後將溶液冷凍乾燥得到產物(NAcHis-DSPE-PEG)。
將DOX．HCl溶於DMSO後再加入triethyl amine於室溫下避光攪拌48小時，將salt form的DOX還原成base form以增進其疏水性進而提升藥物包覆率。而理想化的條件為，triethyl amine的添加量為DOX莫耳數之2倍。
將預先改質完成的NAcHis-DSPE-PEG溶於DMSO之後滴入pH 7.4 phosphate buffer中劇烈攪拌5分鐘後，於攪拌情況下滴入預先溶於DMSO中的PLGA(LA/GA=85/15，2.0mg)及DOX並持續劇烈攪拌30分鐘。而理想化的條件為，DOX的進料量為PLGA之20wt%。
將均勻混和後之溶液於室溫靜置平衡30分鐘後置入透析模(MWCO 12000~14000)中，於4℃下對磷酸鹽緩衝溶液透析一天以移除未包覆之DOX與DMSO即完成其製備，並將DOX-loaded NAcHis-DSPE-PEG/PLGA Nanoparticles簡稱為DOX-loaded NHDPNs。
【第五實施例】
NAcHis-DSPE-PEG之製備如【第四實施例】，本實施例不再贅述。將DOX．HCl溶於DMSO再加入triethyl amine於室溫避光下攪拌24小時後備用，此舉主要是增加DOX的疏水性以提高藥 物包覆效率。而理想化的條件為，triethyl amine的添加量為DOX莫耳數之2倍。
將含有NAcHis-DSPE-PEG的DMSO溶液在磁石攪拌下滴入分散於磷酸緩衝溶液(pH 7.4,0.01M)，轉速設定為1350rpm攪拌五分鐘。再將含有PLGA(LA/GA：85/15)、DOX與ICG的DMSO溶液，緩慢滴入含NAcHis-DSPE-PEG之磷酸緩衝溶液(DMSO/Water(v/v)=1/7)，於避光環境下磁石攪拌30分鐘。而理想化條件為2mg PLGA、0.4mg DOX、0.4mg ICG與0.35_mL DMSO。
將上述溶液裝入透析袋(MWCO 12000~14000)內對磷酸緩衝溶液(pH 7.4,0.01M)透析24小時，以移除未包覆之藥物與DMSO，即可得酸鹼應答型奈米微粒ICG/DOX-loaded NAcHis-DSPE-PEG/PLGA Nanoparticles(簡稱ICG/DOX-loaded NHDPNs)水溶液並將透析後的酸鹼應答型奈米微粒溶液保存於4℃冰箱。
【第六實施例】
NAcHis-DSPE-PEG之製備如【第四實施例】，此實施例不再贅述。本發明另一實施例的酸鹼應答型奈米微粒為首先將DOX．HCl溶於DMSO再加入triethyl amine於室溫避光下攪拌24小時後備用，此舉主要是增加DOX的疏水性以提高藥物包覆效率。而理想化的條件為，triethyl amine的添加量為DOX莫耳數之2倍。
將含有NAcHis-DSPE-PEG的DMSO溶液，在磁石攪拌下滴入分散於磷酸緩衝溶液(pH 7.4,0.01M)，轉速設定為1350rpm攪拌五分鐘。再將含有PLGA(LA/GA：85/15)、DOX與SPION(superparamagnetic iron oxide nanoparticles，SPION)的DMSO溶液，緩慢滴入含NAcHis-DSPE-PEG之磷酸緩衝溶液(DMSO/Water(v/v)=1/7)，於避光環境下磁石攪拌30分鐘。而理想化條件為2.0_mg PLGA、0.4_mg DOX、0.2_mg SPION以及 0.35_mL DMSO。
接著，將上述溶液裝入透析袋(MWCO 12000~14000)內對磷酸緩衝溶液(pH 7.4,0.01M)透析24小時，以移除未包覆之藥物與DMSO，即可得酸鹼應答型奈米微粒(SPION/DOX-loaded NHDPNs)之水溶液並將透析後的酸鹼應答型奈米微粒溶液保存於4℃冰箱中。

Claims (11)

  1. 一種酸鹼應答型奈米微粒，為一酸鹼應答型高分子材料與一聚乳酸聚甘醇酸(PLGA)經自組裝形成，其特徵在於，該酸鹼應答型奈米微粒隨著外部環境pH值由7.4變化至5.0，其表面電位由負電變成正電，該酸鹼應答型高分子材料係由一聚乙二醇衍生物與一組胺酸衍生物(R-Histidine)經化學反應所形成；其中，該酸鹼應答型高分子材料係通過該聚乙二醇衍生物與該聚乳酸聚甘醇酸連接，且該組胺酸衍生物為N-乙醯基組胺酸(N-acetyl-Histidine)、L-組胺酸(L-Histidine)、D-組胺酸(D-Histidine)或3-甲基組胺酸(3-Methyl-L-histidine)。
  2. 如請求項1所述之奈米微粒，其中該酸鹼應答型高分子係由該聚乙二醇衍生物與該組胺酸衍生物(R-Histidine)經酯化反應而形成。
  3. 如請求項2所述之奈米微粒，其中該聚乙二醇衍生物為維他命E(TPGS)或二硬脂基磷脂乙醇胺(DSPE-PEG)。
  4. 如請求項1所述之奈米微粒，其中該酸鹼應答型奈米微粒包含一親水區與一疏水區，該親水區位於相對於該疏水區的外側。
  5. 如請求項4所述之奈米微粒，其中該疏水區進一步包覆抗癌藥物、顯影劑、光熱藥劑、奈米金屬粒子或其組合。
  6. 如請求項1至5中任一項所述之奈米微粒，其中該酸鹼應答型奈米微粒的表面電位為-25~10(mV)。
  7. 一種酸鹼應答型奈米微粒應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途，其包括：(a)一聚乙二醇衍生物與一組胺酸衍生物(R-Histidine)經化學反應所形成一酸鹼應答型高分子材料；以及(b)該酸鹼應答型高分子材料與聚乳酸聚甘醇酸(PLGA)經自組裝形成一酸鹼應答型奈米微粒；其中，該酸鹼應答型高分子材料係通過該聚乙二醇衍生物與該聚乳酸聚甘醇酸連接，且該組胺酸衍生物為N-乙醯基組胺酸(N-acetyl-Histidine)、L-組胺酸(L-Histidine)、D-組胺酸(D-Histidine)或3-甲基組胺酸(3-Methyl-L-histidine)。
  8. 如請求項7所述之酸鹼應答型奈米微粒應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途，其中該聚乙二醇衍生物為維他命E(TPGS)或二硬脂基磷脂乙醇胺(DSPE-PEG)。
  9. 如請求項7所述之酸鹼應答型奈米微粒應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途，其中該酸鹼應答型奈米微粒包含一親水區與一疏水區，該親水區位於相對於該疏水區的外側。
  10. 如請求項7所述之酸鹼應答型奈米微粒應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途，其中該疏水區進一步包含抗癌藥物、顯影劑、光熱藥劑、奈米金屬粒子或其組合。
  11. 如請求項7至10中任一項所述之酸鹼應答型奈米微粒應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途，其中該酸鹼應答型奈米微粒的表面電位為-25~10(mV)。
TW104119949A 2015-06-22 2015-06-22 酸鹼應答型奈米微粒的製備並應用於製備促進抗癌藥物於腫瘤的傳輸與深層滲透之藥物的用途 TWI572369B (zh)

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