開發腫瘤標的性載體應用於蛋白質的胞內遞輸

Development of Tumor Targeted Carriers for Intracellular Protein Delivery

葉紫涵(Tzyy-Harn Yeh)

指導教授 ： 沈麗娟

國立臺灣大學/藥學專業學院/藥學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.11333

摘要

近年來，因為蛋白質活性的專一反應和高效率使蛋白質藥物在癌症治療上的應用越來越具有前景，然而因為蛋白質的親水性和分子大小，若沒有特定的轉運蛋白或載體的幫助很難穿透細胞膜進入細胞，所以大多數應用在癌症治療上的蛋白質藥物都作用在細胞外，包括細胞膜上、血液中和腫瘤細胞外的微環境。隨著對於腫瘤生物學的了解，有更多前瞻性的蛋白質標的可以應用在癌症治療上，但受限於蛋白質對於細胞膜的穿透度低，使這些作用在細胞內的蛋白在臨床應用上有很大的限制，為了增加這些蛋白質藥物的應用性，具有腫瘤標的和胞內遞輸作用的載體的開發是很重要的一項課題。本篇研究利用兩種不同機制的策略來達到腫瘤標的性的蛋白質胞內遞送: 1. 利用標的腫瘤細胞上的受質受體誘發的胞吞作用(ligand-induced receptor mediated endocytosis)；2. 利用酸鹼敏感的穿膜蛋白(pH-sensitive cell penetrating peptide)促進蛋白質在微酸性的環境下進入腫瘤細胞。
第一個部分的研究利用半乳醣化的白蛋白(galactosylated albumin)作為標的肝癌細胞的載體來運送細胞色素c (cytochrome c)。半乳糖可以被肝癌細胞上大量表現的脱唾液酸糖蛋白受體(asialoglycoprotein receptor，ASGPR)辨認，進而誘發胞吞作用把白蛋白及所攜帶的細胞色素c送進細胞內。首先，細胞實驗結果顯示，相較於未經修飾的白蛋白，半乳醣化的白蛋白顯著的提升了進入細胞內的量。接著將細胞色素c用可還原分離的雙硫鍵連結在半乳醣化的白蛋白上，使細胞色素c可以在細胞內從載體上被釋放出來。細胞吞噬實驗和毒性試驗結果顯示，利用半乳醣化白蛋白作為載體可以顯著增加細胞色素c遞送到細胞內的量，並且經由誘發細胞凋亡降低癌細胞的存活率。
第二部分的研究利用酸鹼敏感的穿膜蛋白來攜帶蛋白精氨酸脫亞氨酶(arginine deiminase，ADI)在微酸的腫瘤環境中增加胞內遞送。穿膜蛋白使用的是先前由本實驗室篩選出的HBHAc，接著利用組胺酸(histidine，H)和穀氨醯胺(glutamine，E)重複序列的短片段胜肽(HE)控制酸鹼反應性。本實驗利用蛋白質重組技術製造出帶有HBHAc和HE胜肽的蛋白精氨酸脫亞氨酶(HBHAc-HE-ADI)。利用培養在缺氧環境下的MDA-MB-231乳癌細胞作為胞內遞送及細胞毒性的體外模式。當MDA-MB-231生長在缺氧的環境下時，會誘發對蛋白精氨酸脫亞氨酶的抗藥性，同時因缺氧引起的大量醣解作用會酸化細胞培養液達到微酸性的環境。細胞實驗結果顯示，HBHAc-HE-ADI可針對缺氧酸化的細胞增加胞內遞送，同時具有酸性活化的細胞毒性，並且逆轉了缺氧誘發的抗藥性。此外，於動物實驗中發現，HBHAc-HE-ADI可利用酸鹼敏感性達到腫瘤標的作用，並達到與臨床試驗中的ADI-PEG20相同的腫瘤遞送效果。
本研究中利用兩個不同設計途徑來達到腫瘤標的性的胞內遞送。第一部分顯示利用半乳醣化的白蛋白可有效的增加細胞色素c遞送到肝癌細胞內，同時可誘發細胞凋亡而降低癌細胞存活率。第二部分利用酸鹼敏感性的穿膜蛋白將蛋白精氨酸脫亞氨酶遞送到微酸環境中的癌細胞，結果顯示其具有酸性活化的細胞毒性，同時抑制了細胞的抗藥性，並可於動物實驗中達到腫瘤標的的效果。

關鍵字

白蛋白 ； 細胞色素c ； ?唾液酸糖蛋白受體 ； 胞內遞送 ； 蛋白精氨酸脫亞氨? ； 酸鹼敏感性

並列摘要

The highly specific and effective action of proteins to their targets makes them a promising candidate as therapeutic agents. For the past decades, utilizing proteins therapeutics in cancer treatment became a promising method. Due to the hydrophilic nature and bulky size of proteins, they have low cell permeability without specific transporters or carriers. Therefore, most of the protein therapeutics currently under development or in clinical setting has their targets extracellularly. With the growing knowledge of cancer biology, there is more promising protein therapeutics which may target on the unbalancing molecular pathways inside of the cells. However, this category of protein therapeutics has limited clinical application. In order to improve therapeutic values of proteins acting inside of cells, carriers enhancing intracellular delivery to tumor cells are in needed. In this study, we aimed to achieve tumor targeted intracellular protein delivery through two different mechanisms: 1. ligand-induced receptor mediated endocytosis; 2. pH-sensitive cell penetrating peptide facilitated cellular internalization.
For the first part of the study, we used a galactosylated albumin based carrier for intracellular delivery of cytochrome c (Cyt c) to hepatocarcinoma cells. Galactosylated albumin is recognized by highly expressed asialoglycoprotein receptors (ASGPR) on hepatocarcinoma cells and is further internalized into cells via receptor mediated endocytosis. The cell uptake study showed galactosylation significantly increase albumin internalization in a dose dependent manner. Then Cyt c was chemically conjugated to galactosylated albumin through a reducible disulfide linker in order to release Cyt c from the carrier inside of cells. We tested cellular uptake and cytotoxicity of Cyt c conjugates in ASGPR positive and negative hepatocarcinoma cells. The results showed galatosylated albumin significantly increased cellular uptake in both cell types through a galactose-related endocytic pathway, and resulting in cytotoxicity in a dose dependent manner through induction of apoptosis.
For the second part of the study, we construct a pH-sensitive cell penetrating peptide (CPP) targeting the mildly acidic tumor microenvironment for intracellular delivery of arginine deiminase (ADI). The pH sensitivity of HBHAc, a CPP identified in our laboratory, was controlled by histidine-glutamine (HE)n oligopeptide. The protein constructs were generated by recombinant technology. We create a mildly acidic in vitro model by culturing MDA-MB-231 breast cancer cells in hypoxic condition. MDA-MB-231 developed ADI resistant in hypoxia. Additionally, the intensive glycolysis induced by hypoxia acidified culture media to as low as pH 6.1. The HBHAc-HE-ADI showed an increased cellular uptake and a pH activated cytotoxicity in hypoxic cultured MDA-MB-231 which restored treatment sensitivity. In addition, HBHAc-HE-ADI exhibited a comparable tumor accumulation effect to ADI-PEG20, a PEGylated ADI which is currently in clinical trials for treating different cancers.
In this study, we showed two approaches for tumor targeted intracellular protein delivery. First, galactosylated albumin served as a protein drug carrier for intracellular delivery and intracellular delivery of Cyt c can be an alternative therapeutic strategy. Second, HBHAc-HE-ADI can target to mildly acidic tumor microenvironment and intracellular delivery of ADI restored hypoxia-associate resistance in MDA-MB-231.

並列關鍵字

albumin ； cytochrome c ； hepatocarcinoma ； ASGPR ； intracellular protein delivery ； arginine deiminase (ADI) ； pH sensitive

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