帕金森氏症 (Parkinson’s disease, PD)屬於慢性神經退化性疾病，相較於傳 統的藥物及手術治療，基因治療特有的內生性及長效性使其更適合被用於 PD 的治療。然而由於基因片段 (plasmid DNA, pDNA)的在體循環的過程中其穩定 性差且無法有效地進入細胞核產生轉染的效果，因此適當的基因載體 (Gene vector)在基因治療中扮演關鍵的角色。傳統的基因載體多以病毒為主，病毒擁 有非常良好的轉染效果，藉由感染的方式使宿主細胞大量產生特定蛋白，然而 安全性及高成本大幅地限制其應用發展。相反地，非病毒載體雖具備安全性及 易被製備等優點使其擁有高度的潛力，然而其轉染效果卻遠低於病毒式載體。 在非病毒式載體將 pDNA 遞送進細胞核的過程會遇到來自細胞胞器的阻礙導 致轉染率下降，阻礙包含:(1)細胞質:細胞質是由雙層磷脂質組成的親脂層，一 般外來物質藉由胞吞 (endocytosis)的方式進入細胞，然而胞吞所需的時間往往 要長達數個小時，導致 pDNA 等親水物質無法有效地進入細胞; (2)溶酶體系統: 溶酶體內含有大量酸性水解酵素，其原功能是用於保護細胞免於外來物質的侵 入，然而在基因遞送的過程中 pDNA 容易被溶酶體分解導致轉染效果大幅下 降; (3)細胞骨架:pDNA 於細胞內的輸送是藉由運動蛋白 (motor protein)的幫助 下沿著細胞骨架而進到細胞核，然而錯綜復雜的細胞骨架結構導致 pDNA 無法 有效地進到細胞核進行轉染，此外在治療腦內疾病時還必須考慮血腦屏障的因 素。本研究提出複合式的基因載體以突破基因遞送的阻礙，利用 SSPEI-SPIO (PSPIO)與 pDNA 以縮合的方式相接形成 PSPIO-pDNA，接著再將此複合材料 掛載至微氣泡 (microbubbles, MBs)表面 形成具備聲學及磁學特性的 PSp-MBs 複合式基因載體。藉由聲學穴蝕效應及磁導引的效果可以有效地突破細胞膜的 阻礙將 PSPIO-pDNA 遞送入細胞質中，接著再靠著 SSPEI 產生的海綿質子效應幫助逃離溶酶體的分解，最後再靠著二次磁場使細胞骨架排列一致使 pDNA 更有效率地遞送至細胞核。
本研究成功利用電性吸附的方式以 3.61 pg/MB 的掛載效率將 PSPIO-pDNA(191±2.26 nm)掛載至 MB (1.18±0.04 μm)上，並以 pGFP 與 pGDNF 分別 利用SH-sy5y 細胞株進行細胞實驗驗證其基因轉染效率與MitroPark帕金森氏 症動物模型的治療。在細胞實驗中首先同時利用超音波(frequency = 1 MHz, pressure = 0.3 MPa, duty cycles = 0.5 %, sonication duration = 1 min)及磁導引 (0.37 T, 20 mins)將 PSPIO-pDNA 送入細胞質中，接著將細胞於細胞培養箱放置 24 小時等待海棉質子效應及 PSPIO 與 pDNA 分離後，再利用二次磁場 (0.37 T, 60 mins)使 pDNA 更有效率地遞送至細胞核，再經過 24 小時後以流式細胞 儀定量轉染結果。另外，本研究紀錄老鼠行走過 80 公分之獨木橋所需的時間 及老鼠於開放空間行走的總距離來觀察老鼠之運動能力與意願並利用西方墨 點分析法以驗證帕金森氏症的治療效果。
根據轉染定量結果，本研究提出的基因轉染平台中超音波與磁導引分別提升 1.49 倍與 1.50 倍，而同時施打超音波並配合磁導引時則能提升 2.45 倍且基因 轉染效率達到 16.35±0.46 %，而在加入二次磁場後則能將轉染效率提升至 22.73±6.33 %，是目前商用轉染試劑 TransIT®-LT1 的 2.2 倍。此外，在帕金森 氏症的治療當中，經過治療的老鼠可以分別提升 1.5 倍的平衡能力及 3.06 倍 的運動能力，且在西方墨點分析法中可以提升 2.1 倍的 TH+含量，代表老鼠在 經過治療過後可以有效提升合成多巴胺的效率。本研究提出超音波結合磁場之 基因遞送平台，藉由 PSp-MBs 本身表現之聲學及磁學特性下，搭配超音波與 磁場可以有效地開啟血腦屏障並突破非病毒式載體在基因遞送上的限制，有效 地將 pDNA 遞送至細胞核達到轉染效果，此外本研究成功遞送 pGDNF 應用於 帕金森氏症的治療，未來此平台可結合其他基因片段並用於其他神經退化性疾 病的治療。

Genetic treatment for Parkinson’s disease (PD) by plasmid glial cell-line derived neurotrophic factor (pGDNF) has the potent trophic effect on damaged dopaminergic neurons. Ultrasound (US)-based plasmid DNA (pDNA) delivery is a promising mean for central nervous system treatment. However, three barriers including, (1) cell membrane reducing intracellular pDNA uptake, (2) lysosome degrading the delivered pDNA and (3) cytoskeleton lowering pDNA entry into nucleus, hamper pDNA transfection and expression (typically 4-10 %). This study proposed the polyethylenimine (PEI)-superparamagnetic iron oxide-pDNA loaded microbubbles (PSp-MBs) in conjunction with US and two-step magnetic force to increase the efficiency of gene delivery. In other words, the intracellular accumulation of PSp complexes was enhanced by concurrently performing US (cavitation) and step I magnetic force (magnetic effect). Secondly, the PEI allowed PSp complexes escaping from lysosome degradation (proton sponge effect). In final, the amount of pDNA entering nucleus could be increased by step II magnetic-mediated cytoskeletons re-organization (magnetic effect).
The PSp-MBs (3.61 pg PSp/MB) were prepared by loading PSp (150±5.4 nm) onto the lipid-shell of MBs (1.18±0.04μm) by electrostatic force. The plasmid green fluorescence protein (pGFP) and pGDNF were used to quantify the efficiency of gene delivery and repair dopaminergic neurons in SH-sy5y neuron-like cell and genetic PD mice (MitoPark), respectively. The PSp delivery was carried out by FUS (frequency = 1 MHz, pressure = 0.3 MPa, duty cycles = 0.5 %, sonication duration = 1 min) and step I magnetic force (0.37 T, 20 mins) with PSp-MBs. After lysosome escape, the step II magnetic force (0.37 T, 60 mins) was applied to the right side of cells for pDNA transporting into nuclear. To verify the in vivo treatment outcome, the time for crossing an 80 cm-beam and the walking distance in open field by PD’s mice (N=30) were recorded weekly to trace the motor balance and willingness, respectively.
The gene transfection rate in the proposed system was 2.2 fold higher than that of the commercial agent (TransIT®-LT1). The transfection rates could be boosted up ~11%, ~7%, and 2% by cavitation-magnetic hybrid enhanced cell membrane permeabilization, proton sponge effect and magnetic-assisted cytoskeleton- reorganization, respectively. In vivo data suggested that the system improved 1.5 and 3.06 folds of balance ability and motor ability compared to untreated PD mice, individually. This study proposed a novel US-magnetic hybrid gene delivery platform and potentially could be integrated with other therapeutic genes for treating neurodegenerative diseases in future.

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