經鼻腔至腦部藥物遞送：作用機轉與超音波促進藥物傳輸探討

Abstract

血腦障壁為藥物遞送至腦部的主要障礙，限制多數藥物的進入。鼻腔給藥（Intranasal, IN）為一種有潛力且非侵入性的方式，能避開血腦障壁經由嗅神經及三叉神經途徑，直接傳遞藥物至腦部。近年來，關於膠淋巴系統的研究顯示，血管周圍腔隙（perivascular space, PVS）可能在藥物於腦內快速分布中扮演關鍵角色。但是目前尚無研究探討IN藥物遞送腦部的不同時間點與腦內分佈以及與PVS的關係。此外，關於使用低強度超音波促進IN藥物傳遞至腦部的證據亦仍有限。因此，我將透過兩個主要的研究主題，探討鼻腔至腦部藥物遞送的機制及應用超音波增強IN遞送腦部的效率。 在第一部分研究中，我們使用追蹤劑 Fluoro-Gold (FG)，分別以IN給藥及直接嗅球注射於小鼠模型中追蹤藥物動態。我們發現在IN給藥後30分鐘內，FG可迅速到達大腦皮層與三叉神經，並隨時間沿PVS深入腦內，然而在其他腦區並未見到此現象。免疫螢光染色分析顯示IN給藥後FG與星狀膠質細胞標記物的共定位現象，提示IN給藥主要藉由細胞外傳輸及PVS達成廣泛分布。相較之下，直接嗅球注射後FG的傳輸速率較慢且分布範圍較窄，且FG與星狀膠質細胞標記物並未出現共定位現象，反映出細胞外路徑於IN傳遞中的重要角色，我們也證實FG在動物模型中是評估經鼻腔藥物遞送模型的可靠追蹤劑。 第二部分研究中，將探討低強度經顱超音波刺激，在無搭配微泡下，是否可促進鼻腔藥物遞送。首先我們在活體外透過測量聲壓以測定此客製化低強度超音波系統經顱骨傳導後的聲壓衰減程度。接著我們使用10-kDa螢光標記Dextran作為示蹤劑，IN給藥後施加超音波刺激。我們發現在4小時追蹤中，IN結合超音波（IN+US）組在嗅球及三叉神經中的藥物濃度顯著高於IN單獨組及假處理組。共軛焦顯微鏡的分析顯示，藥物主要聚集於PVS，並且組織學檢查未見微出血或腦組織損傷，證實超音波刺激在本研究條件下能達到促進藥物遞送的效果且具有良好安全性。 綜合上述兩個主要研究的結果，我們確認了鼻腔給藥主要利用細胞外路徑並依賴膠淋巴系統透過PVS以促進腦內分布。此外，我們也證實了低強度超音波能在不搭配微泡的情況下，安全並有效地提升鼻腔至腦部藥物傳遞效率。此結果不僅深化了IN藥物遞送腦部機制的理解，也為未來開發非侵入性腦刺激的治療與藥物遞送提供了重要實證基礎。

The blood-brain barrier (BBB) constitutes a substantial challenge for the effective transportation of therapeutic agents to the brain. Nasal administration offers a promising non-invasive pathway to bypass BBB via the olfactory and trigeminal nerve routes. Recent discoveries regarding the glymphatic system suggest that the perivascular space (PVS) may be essential for facilitating the rapid distribution of drugs thorough the brain. However, no studies to date have visualized the PVS or trigeminal nerve at different time points to elucidate their involvement in IN delivery pathways. Moreover, evidence supporting the use of low-intensity ultrasound to facilitate IN drug delivery to the central nervous system (CNS) remains limited. Therefore, this thesis aims to investigate the mechanisms and potential enhancement strategies of nasal-to-brain drug delivery through two main studies. In the first study, we used Fluoro-Gold (FG), a retrograde neuronal tracer, to evaluate intracellular and extracellular transport following IN administration and direct transcranial injection to the olfactory bulb using the mouse model. Whole-brain and brain section imaging revealed that FG rapidly reached the outer cortex and trigeminal nerves within 30 minutes post-IN delivery and progressively penetrated deeper brain regions over time, primarily along PVS. Immunofluorescence staining confirmed FG co-localized with astrocytes, suggesting that extracellular transport via the glymphatic system is critically involved in IN-mediated delivery to brain. In contrast, direct olfactory bulb injection resulted in slower and more restricted transport, with no observed co-localization between FG and astrocytes, further emphasizing the critical role of the extracellular pathway in IN drug delivery. FG demonstrated high utility as a reliable tracer for assessing the IN drug delivery model in mice. The second study assessed whether low-intensity transcranial ultrasound, applied without microbubbles (MBs) contrast agents, could enhance IN delivery efficiency. The custom-made low-intensity ultrasound system was evaluated under ex vivo conditions by measuring acoustic pressure to determine the extent of transcranial attenuation. Afterward, a fluorescent 10-kDa dextran tracer was administered intranasally, followed by planar ultrasound treatment across the entire brain. Quantitative analysis demonstrated that at 4 hours post-treatment, the IN combined with ultrasound (IN+US) group showed significantly higher dextran concentrations in the olfactory bulb and trigeminal nerves compared to the IN-only and sham groups. Confocal microscopy revealed dextran accumulation predominantly along PVS structures. Histological examination confirmed no detectable evidence of microhemorrhage or cerebral tissue injury, demonstrating that ultrasound with low-intensity is a safe adjunct to enhance IN drug delivery. Together, these findings highlight that nasal-to-brain delivery predominantly relies on extracellular pathways and the glymphatic system through PVS for efficient distribution. Moreover, we provide the first experimental evidence that low-intensity ultrasound without MBs can safely augment IN drug delivery. These insights offer new directions for the development of non-invasive therapeutic stimulation targeting neurological diseases.