由兩性高分子(amphiphilic polymer)，如雙團聯共聚物(diblock copolymer)及三團聯共聚物(triblock copolymer)所形成的核殼結構(core-shell structure)在高分子微胞藥物傳輸系統(polymeric micellar drug delivery system)應用上具有很多特殊性質與優點。藉由高分子微胞的包覆能夠增進疏水性藥物於水溶液的溶解性及體內的生物可利用度(bioavailability)。高分子微胞外層的殼結構(shell)可由為生物可相容性(biocompatible)之親水性高分子所組成，其能避免網狀內皮組織(reticuloendothelial system)的吸收。而其內層的核結構(core)能包埋或鍵結疏水性藥物保護疏水性藥物。此外，高分子微胞為奈米尺寸(nano-scale)能夠避免被身體代謝系統所排除且在體內能有長時間的循環。
以聚□唑啉高分子poly(2-ethyl-2-oxazoline)(PEtOz)為親水性鏈段再搭配聚酯類之聚乳酸高分子poly(L-lactide)(PLLA)為疏水性鏈段構成本研究中的兩性高分子。其乃藉由開環聚合反應合成聚□唑啉/聚乳酸雙團聯共聚物(PEtOz / PLLA diblock copolymer)與聚□唑啉/聚乳酸三團聯共聚物(PEtOz / PLLA triblock copolymer)。利用兩性高分子在水溶液中自我組裝(self-assembly)行為製備成高分子微胞。本研究中的高分子微胞具有環境應答性(stimuli-sensitive)，如溫度敏感性(thermo-sensitive)與酸鹼敏感性(pH-sensitive)的特性以應用在癌症治療上。
本研究之藥物傳遞方式主要是在細胞內的藥物釋放上。以靜脈注射方式使高分子藥物載體進入體內，藉由Enhanced Permeability and Retention effect (EPR effect效應)累積於腫瘤組織後，再利用胞飲作用(endocytosis)使高分子藥物載體吞噬進入到細胞內。因細胞內pH值的改變使高分子藥物載體的結構產生破壞釋放出藥物達到癌症治療的效果。本研究所設計的聚□唑啉/聚乳酸團聯高分子微胞(PEtOz/PLLA block copolymer micelle)能夠在生理環境(pH7.4)下穩定地保護藥物，然而在細胞內由於吞噬小體(endosomes)或溶酶體(lysosomes)所造成的pH值的下降(pH 4~5)，使高分子藥物微胞變形破壞釋放出藥物。
所製備的聚□唑啉/聚乳酸團聯高分子微胞其粒徑分佈小於200 nm。利用動態光散射(dynamic light scanning)探討微胞之聚集行為、酸鹼敏感性質及微胞於牛血清蛋白(bovine serum albumin)之穩定性。並以螢光光譜儀(fluorescence spectrophotometer)探討微胞之臨界微胞濃度(critical micelle concentration)及微胞結構破壞等性質。抗癌藥物(Doxorubicin , Dox)以物理性作用力包覆於高分子微胞內的疏水性聚乳酸(PLLA)之內核。以聚□唑啉/聚乳酸三團聯共聚物所形成之奈米微胞其藥物包覆率達30 % 以上；聚□唑啉/聚乳酸雙團聯共聚物所形成之奈米微胞其藥物包覆率達20 %以上。在37℃下，兩種不同團聯共聚物所形成之奈米微胞在酸性環境下(pH5.0)皆具有快速釋放藥物之特性。在初期3小時內釋放量皆高於30 % 而在中性環境下(pH7.4)由於奈米微胞結構穩定，使藥物釋放速率十分緩慢。並利用人類子宮頸癌細胞(HeLa)與藥物微胞進行細胞培養，經MTT比色分析，證明這種抗癌藥物微胞的確具有抑制人類子宮頸癌細胞(HeLa)增生繁殖並有效毒殺癌細胞之效果，而達到癌症治療的效果。
本研究中，PEtOz / PLLA團聯共聚物兼具環境應答性、生物可分解性與低毒性等優點，將其應用於智慧型奈米藥物微胞的設計上，體內與體外之藥物釋放情況皆非常優異。以共軛焦顯微鏡影像證明微胞結構在細胞酸性胞器內被破壞，藥物能成功地在細胞質之酸性胞器內被釋放，達到適時、適地之控制釋放的效果。綜合上述結果可知，此包埋抗癌藥物(Doxorubicin, Dox)之聚□唑啉/聚乳酸團聯高分子微胞在細胞內之藥物傳輸上有優良的效果，顯示此一團聯高分子微胞在藥物傳輸上有極大的潛力。

Abstract
Polymeric micellar drug delivery systems (MDDSs) of core-shell architecture based on amphiphilic AB diblock or ABA triblock copolymers possess numerous advantages. They improved solubility and bioavailability of hydrophobic drugs that were poorly soluble or insoluble in water. Micelles with biocompatible hydrophilic shell exhibited low uptake by the reticuloendothelial system even if they had a nonbiocompatible core and hydrophobic core significantly protect the incorporated drug . Additionally, polymeric micelles with nano-scale could avoid the recognition by MPS or RES and they prolonged the circulation time in blood.
In this study, polymeric micelle with environmental stimuli-sensitive properties such as thermo-sensitive properties and pH-sensitive properties were prepared for used in drug delivery. Polymeric micelles were self-assembled from diblock and triblock copolymers in aqueous solution, respectively. Amphiphilic block copolymers with hydrophilic block segment of poly(2-ethyl-2-oxazoline)(PEtOz) and biodegable aliphatic polyester segment of poly(L-lactide)(PLLA) were synthesized from cationic ring-opening polymerization.
This study proposes a new delivery system, which was potentially useful for targeted drug delivery, and rapidly changes the micelle structure in response to changes in intracellular pH. Following intravenous administration, polymeric micelles were accumulated in tumor tissue by EPR effect, and then taken up to cells via endocytosis process. The pH value of endosomal comportments were decreased from 7.4 to 5 because protons are pumped into the vesicles. From our design, the micellar structures can stabilized to preserve hydrophobic drug(Doxorubicin, Dox) under physiological conditions(pH7.4) and selectively release the drugs by sensing the intracellular pH change in endosomes and lysosomes(pH 4~5).
The micelles were self-assembled from PEtOz-PLLA diblock amphiphilic copolymers and PLLA-PEtOz-PLLA triblock amphiphilic copolymers, respectively. The hydrophobic anticancer drug could be entrapped into the hydrophobic core resign of micelles. The characterizations, pH-sensitive properties, micellar stability were determinated by dynamic light scanning(DLS). Additionally, critical micellar concentration(CMC) and structure changed properties of micelle were determinated by fluorescence spectrum.
The mean diameters of micelles were all less than 200 nm and exhibited low polydispersity index. The drug loading level of Doxorubicin(DOX) incoporated into the PEtOz/PLLA triblock polymeric micelles can be as high as 30 % . The drug loading level of Doxorubicin(DOX) incoporated into the PEtOz/PLLA diblock polymeric micelles can be as high as 25 % . At 37℃, the cumulated released rate of Dox from PEtOz/PLLA diblock polymeric micelle at pH5.0 was about 35 % in the initial 3 hour. On the other hand, both micelles exhibit less drug relesed at pH7.4 , indicating that micelles were stabilized in physiological conditions. In use of the method of MTT assay, we could confirm the inhibiting proliferation of HeLa by the releasing anticancer drug and effective therapy to cancer in vitro.
In conclusion, PEtOz /PLLA block copolymers possessed the advantages of environmental sensitivity, biodegradability and lower cytotoxicity, that were suitable for used in drug delivery, especially for cancer therapy. The drug released behaviors of micelles incorpotated with Dox were excellent either in buffer solution or in intracellular endosomal compartment. The result of CLSM observation indicated the release of drug successfully released in the acidic organelles due to the deformation of the micelle structure. As the results, these micelles loaded anticancer drug(Dox) have great potential for application in drug delivery system, especially for used in intracellular drug delivery .

Keywords: Poly(2-ethyl-2-oxazoline)(PEtOz) , Poly(L-lactide)(PLLA), block polymer, micelle, core-shell, stimuli-sensitive, biodegable , self-assembly, endocytosis process.,cancer therapy, intracellular drug delivery

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