聚乙二醇(poly(ethylene glycol), PEG)接枝聚乙烯亞胺(polyethylenimine, PEI)與基因所組合成的複合體，由於低毒性，值得發展為用於基因治療上之基因載體。不過此複合體在細胞或者活體實驗上卻顯現出較低轉染能力，故此複合體常需要加入具標靶能力的配位體(ligand)以增加其細胞結合及轉染能力。整合素(integrin)，αvβ3在腫瘤新生血管上扮演著重要的角色，已成為許多腫瘤診斷及治療之標靶。而精胺酸-甘胺酸-天門冬胺酸(Arg-Gly-Asp, RGD)胜肽序列與αvβ3受器具有高度特異性親和力。在過去的研究中，探測標靶型基因傳輸載體的標靶能力往往依賴於植入報導基因的表現，而在本研究中，我們嘗試標誌加馬射線放射性核種在標靶型基因載體上，利用單光子發射斷層造影/電腦斷層造影(single photon emission computed tomography, SPECT/computer tomography, CT)影像評估此載體在一動物模式之生物分佈及標靶能力。
本研究製備一鍵結有雙環型雙分子RGD, E[c(RGDyK)]2的PEG-g-PEI高分子基因載體，並予以標誌鎝99m作為放射性追蹤劑應用到活體生物分佈實驗。所配製三種PEG-g-PEI產物的PEG與PEI的接枝比例為1:1、3:1及10:1，依序分別命名為PP1、PP3及PP10，並以核共振氫譜測定產物接枝比例。為了探討不同PEG與PEI接枝比例之複合體攜帶DNA的能力，分別予以評估不同高分子/去氧核醣核酸之重量比(簡稱P/D)所形成複合體的高分子與DNA之組合能力以及表面電荷。在P/D=1時所有組合的複合體粒徑大小約為90-130 nm，DNA能完善地受高分子保護，其表面電荷電位(zeta potential)呈現40-50 mV。
使用不同PEG/PEI比例的PEG-g-PEI/DNA複合體並在逐漸提升PEG-g-PEI濃度下探討細胞毒性。在任一PEG-g-PEI濃度下，可以觀察到PEG與PEI接枝比率為10的複合體(簡稱PP10/D)表現最低的毒殺性，而不具PEG之複合體PEI/DNA表現最高的毒殺性。在轉染實驗上PP10/D在PP10濃度超過0.025 mg/ml時表現顯著的轉染能力。故在接下實驗選用PP10執行進一步的標靶研究。E[c(RGDyK)]2接合PP10/D 之標靶複合體, RPP10/D比起沒有標靶的複合體PP10/D具有顯著提升1.25倍以上的細胞結合能力及轉染能力。在鎝99m標誌上,PP10/D及RPP10/D兩者均具有高達95%的標誌效率，兩類複合物在食鹽水及大鼠血漿下經24小時皆仍具有高達80%的標誌穩定度。在活體SPECT/CT造影上，可以觀察到在大部分的時間點上RPP10/D在腫瘤位置的吸收較PP10/D為高。利用SPECT影像圈選具相關性的區域(regions of interest, ROI)評估出RPP10/D在腫瘤與肝臟之比值和腫瘤與血池(心臟器官)之比值均較PP10/D為高。在傳統生物分佈實驗結果亦印證SPECT上ROI所分析的生物分佈趨勢。在螢光顯微鏡下觀察到所攜帶的綠螢光蛋白基因(plasmid EGFP)在腫瘤組織上表現出螢光。
由本研究結果顯示，E[c(RGDyK)]2鍵結PEG-g-PEI高分子基因載體，並以99mTc標誌，在植有Hela腫瘤小鼠模式測試證實有潛力成為一個具有標靶且影像導引的基因治療藥物。

The polyplexes of polyethylene glycol (PEG) grafted polyethylenimine (PEI) and conjugated with gene can be used as a gene carrier in gene therapy for its low toxicity. However, the polyplexes displayed lower transfection capability in vitro and in vivo in the past studies. Modification of the polyplexes by binding a targeting ligand may improve the transfection capability. Integrin αvβ3 plays a critical role in tumor angiogenesis and becomes a promising diagnostic and therapeutic target for various solid tumors. It is well known that Arg-Gly-Asp (RGD) peptide has high affinity with αvβ3 integrin. The studies of targeting ability of a gene delivery vehicle usually rely on a design by expression of reporter gene in vivo. In this study, we have attempted to label a targeting gene delivery vehicle with a gamma emitter for evaluating its biodistribution and targeting ability by single photon emission computed tomography/computed tomography (SPECT/CT).
The scope of this study was to prepare targeting polymeric gene carriers based on dimeric cyclic RGD binding PEG-g-PEI (abbreviated as E[c(RGDyK)]2- PEG-g-PEI) and labeling with 99mTc for in vivo study. We have prepared the PEG-g-PEI products with PEG to PEI ratios of 1 to 1, 3 to 1, and 10 to 1, abbreviated as PP1, PP3 and PP10, respectively. The PEG to PEI ratios in the products were confirmed by 1H-NMR. Furthermore, the PEG-g-PEI/DNA polyplexes with different polymer/DNA weight ratios (abbreviated as P/D) were prepared and their surface charges and zeta potentials and formation abilities with DNA were measured. The particles sizes of the PEG-g-PEI/DNA polyplexes were measured to be 90-135 nm. The zeta potential of the polyplexes was measured to be 40-50 mV. The polyplexes had high formation ability with DNA in P/D ratio at 1:1.
We compared the cytotoxicities of PEI/DNA and PEG-g-PEI/DNA fabricated at different PEG/PEI ratios and different P/D ratios. The polyplex of PEG-g-PEI/DNA fabricated with PEG/PEI ratio at 10: 1 and P/D=1, i.e., PP10/D, showed lowest cytotoxicity whereas PEI/DNA showed highest cytotoxicity. In the transfection studies, PP10/D with PP10 concentration at 0.025 mg/ml displayed a highest transfection efficiency in comparison with the other two polyplexes. The RPP10/D polyplex demonstrated significantly higher binding affinity and transfection efficiency than non-targeting PP10/D. Both PP10/D and RPP10/D had high radiolabeling efficiency at greater than 95% and borne with radiochemical stability above 80% either in saline and in rat plasma when stored for 24 h. In in vivo SPECT/CT studies, it was found from the images that RPP10/D presented higher uptake in the tumor than PP10/D through all the postinjection times studied. From the analyses on the regions of interest (ROI) by the SPECT/CT imaging, RPP10/D also showed higher tumor to liver and tumor to blood ratios than PP10/D. The conventional biodistribution study presented corresponding results and confirmed the targeting effect of RPP10/D. The plasmid EGFP (enhanced green fluorescent protein) of RPP10/D for gene transfection was also observed to successfully transfect into the tumor site by fluorescence microscope in this study.
In conclusion, E[c(RGDyK)]2 bound PEGylated PEI complex is a potential gene delivery vehicle as a targeting and imaging agent for gene therapy as evidenced by the study with the Hela tumor bearing mice model.

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