設計腫瘤靶向四苯乙烯基光敏劑以提升肝癌光動力治療效能

Abstract

肝癌為全球癌症相關死亡的主要原因之一，2020年時其發病率居第六，死亡率則高居第三，其中約90%為肝細胞癌（HCC），且病死率超過90%。儘管早期HCC可透過手術切除或肝臟移植獲得治癒，但多數患者確診時已屬中晚期，導致現有療法效果有限。近年來，免疫檢查點抑制劑（ICIs）等免疫療法雖展現延長存活的潛力，但HCC因具備高度免疫耐受性、免疫抑制性微環境及腫瘤異質性，導致療效不一甚至產生抗藥性。在此背景下，光動力治療（Photodynamic Therapy, PDT）作為一種非侵入性、可精確控制時空作用的治療策略，日益受到關注並被視為肝癌潛在的替代療法。本研究旨在開發具備肝癌細胞雙重靶向與高ROS產能的四苯乙烯（tetraphenylethylene, TPE）衍生光敏劑，以提升PDT在肝癌治療中的效果與選擇性。 為克服上述挑戰，本研究設計並合成一系列具給體–π–受體（donor-π-acceptor, D–π–A）結構的四苯乙烯衍生光敏劑，期望兼具高效ROS生成與細胞器/細胞雙重靶向功能。首先，進行核心光敏劑骨架的篩選，透過分子設計導入扭曲內部電荷轉移（twisted intramolecular charge transfer, TICT）機制，以TPE為電子給體，連接N 甲基乙烯基吡啶鎓（N-methyl vinylpyridinium, NMVP）作為受體，構築出六種異構核心（CPM、CMM、COM、GPM、GMM、GOM）。光物理性質分析顯示，所有衍生物在不同溶劑中均展現明顯溶劑變色性與顯著Stokes位移，驗證其TICT行為；ROS偵測結果（EPR、DCFH-DA、DHR-123與SOSG）則確認其主要以Type I機制產生活性氧，其中GPM具最快速的ROS生成能力。 接著在細胞攝取與亞細胞靶向的應用上，選定表現最優的GPM為核心進行功能化修飾：首先去除短鏈PEG5並轉換為酚基手柄，一端接枝N 乙醯 D 半乳糖胺（N-acetyl-D-galactosamine, GalNAc）以介導asialoglycoprotein receptor (ASGPR)專一性攝取，另一端則分別接入三苯基膦（triphenylphosphonium, TPP）靶向線粒體，及對甲苯磺醯胺（p-toluenesulfonamide, NTs）或五氟苯基（pentafluorophenyl, PFP）靶向內質網，成功合成具雙重靶向能力之TPPG、TsG與FBG三種衍生物。 肝細胞癌細胞HepG2體外光毒性實驗顯示，TsG在低劑量光照與低濃度條件下即能有效誘導細胞死亡，且在暗處幾無毒性，展現出高度選擇性與良好安全性。雖然共聚焦雷射掃描顯微鏡（CLSM）尚未觀察到明確的胞器定位，但Gaussian 16下之CAM B3LYP/6 31+G(d,p)理論模擬指出，導引基團可能因分子摺疊產生π–π堆疊效應，進而調控激發態性質並提升系統間窄合（ISC）效率，加強ROS生成。 綜上所述，本研究成功開發具高ROS生成效率與肝癌細胞雙重靶向能力的TPE衍生光敏劑，為肝癌光動力治療提供一項具潛力的創新策略。

Liver cancer remains one of the leading causes of cancer-related mortality worldwide. In 2020, it ranked sixth in incidence and third in cancer-related deaths, with hepatocellular carcinoma (HCC) accounting for approximately 90% of cases and a case-fatality rate exceeding 90%. Although early-stage HCC may be curable through surgical resection or liver transplantation, most patients are diagnosed at intermediate or advanced stages, limiting the efficacy of current treatment options. In recent years, immunotherapies, particularly immune checkpoint inhibitors (ICIs), have shown promise in prolonging survival. However, the intrinsic immune tolerance, immunosuppressive tumor microenvironment, and high tumor heterogeneity of HCC often lead to variable responses and resistance to ICIs. Against this backdrop, photodynamic therapy (PDT) has gained increasing attention as a non-invasive and spatiotemporally controllable treatment modality, emerging as a promising alternative for HCC. This study aims to develop tetraphenylethylene (TPE)-derived photosensitizers with dual targeting capability toward hepatocellular carcinoma cells and high ROS-generating efficiency, thereby enhancing the therapeutic performance and selectivity of PDT in liver cancer treatment. To overcome these challenges, we designed and synthesized a series of TPE-derived photosensitizers featuring donor-π-acceptor (D-π-A) structures, aiming to achieve both efficient ROS generation and dual targeting of cells and organelles. Initially, core photosensitizer scaffolds were screened through molecular design incorporating a twisted intramolecular charge transfer (TICT) mechanism, using TPE as the electron donor and N-methyl vinylpyridinium (NMVP) as the acceptor, resulting in six isomeric cores (CPM, CMM, COM, GPM, GMM, GOM). Photophysical analyses revealed pronounced solvatochromism and large Stokes shifts in various solvents, indicating strong TICT behavior. ROS detection assays (including EPR, DCFH-DA, DHR-123, and SOSG) confirmed that these compounds primarily generate ROS via a Type I mechanism, with GPM exhibiting the most rapid ROS generation. For targeted cellular uptake and subcellular localization, GPM, identified as the optimal core, was further functionalized: the short PEG5 chain was removed and replaced with a phenol handle. One end was conjugated with N-acetyl-D-galactosamine (GalNAc) to mediate specific uptake via the asialoglycoprotein receptor (ASGPR), while the other end was modified with triphenylphosphonium (TPP) for mitochondrial targeting or either p-toluenesulfonamide (NTs) or pentafluorophenyl (PFP) for endoplasmic reticulum targeting, leading to the successful synthesis of three dual-targeting derivatives: TPPG, TsG, and FBG. In vitro phototoxicity assays on HepG2 cells demonstrated that TsG effectively induced significant cell death under low light and low concentration conditions, with negligible dark toxicity, indicating excellent selectivity and safety. Although confocal laser scanning microscopy (CLSM) did not show clear subcellular localization, theoretical simulations using Gaussian 16 at the CAM-B3LYP/6-31+G(d,p) level suggested that the organelle-targeting moieties may influence the excited-state properties through intramolecular π-π stacking caused by molecular folding, thereby enhancing intersystem crossing (ISC) efficiency and boosting ROS production. In conclusion, this study successfully developed TPE-derived photosensitizers with high ROS-generating efficiency and dual targeting capability toward hepatocellular carcinoma cells, offering a promising and innovative strategy for liver cancer photodynamic therapy.