聚焦式超音波開啟小鼠黑質區血腦屏障：動脈與靜脈 藥物灌注路徑之差異探討

Abstract

血腦屏障 (blood-brain barrier, BBB) 結構基礎是具有緊密連接的毛細血管內皮細胞，可以有效防止分子擴散改變正常的腦功能。在維持高度精確的大腦微環境內支持神經元活動方面發揮著至關重要的保護作用。但通過輸送治療性納米粒子治療中樞神經系統疾病的有效性也受到了限制。聚焦式超音波 (Focused Ultrasound, FUS) 結合微氣泡 (Microbubbles, MBs) 開啟 BBB 作為一種新穎的開啟方式。其具有非侵入性，副作用小，可暫時開啟等優點。是未來腦部基因或藥物遞送之理想工具。然而過往微氣泡灌注有經過靜脈藥物灌注 (Intra-Venous, IV) 以及頸動脈藥物灌注 (Intra-Artery, IA) 二種方法，現今研究似乎未有詳細討論此二種微氣泡灌注途徑在開啟效能與全身藥物分布方面的綜合探討。在本研究中我們使用 FUS 開啟 BBB 來探討經由 IV 和 IA 藥物灌注途徑之差異使用小動物模型，並以伊文斯藍染劑 (Evans blue dye, EB) 作為經 IV 或 IA 灌注途徑的治療替代分子。以 EB 滲透濃度和免疫螢光染色來評估 FUS 開啟 BBB 的程度。與單獨使用 MBs 相比，FUS 結合 2.8 × 10^8 MBs/kg 的微氣泡濃度條件下顯著提高 IV(p = 0.00318) 或 IA 途徑 (p = 0.0006) 傳遞的 EB 滲透效率。並且在相同的微氣泡濃度下，IA 途徑的 EB 滲透效率高於 IV 途徑 (p = 0.0276)。將微氣泡濃度增加到 1.12 × 10^9 MBs/kg 進一步提高了 IV (p = 0.0023) 和 IA 組 (p = 0.0008) 的 EB滲透效率，IA 途徑達到了最高的 EB 滲透效率。另外還對不同 FUS 刺激時間進行了探討。其次進行了免疫螢光檢查，對 BBB 通透性的標誌物 IgG 進行亮度量化，在黑質 (Substantia nigra, SN) 腦區發現在高濃度微氣泡情況下，IA 組 BBB 開啟程度優於 IV 組。對 IV 與 IA 實驗組別的安全性也進行了評估，在相同實驗條件下IA 各實驗組別腦組織出血情況均明顯少於 IV 實驗組別。並且 IA 灌注相對 IV 灌注在全身藥物分佈方面也有更低的表現。綜上所述，我們顯示使用 IA 灌注藥物情況下，FUS 開啟 BBB 在增強靶向遞送至 SN 腦區優於 IV 灌注。藥物灌注途徑的選擇會影響 FUS 開啟 BBB 的效率，這對於後續優化神經疾病的基因/藥物傳遞可能至關重要。然而藥物灌注途徑如何影響 FUS 開啟 BBB 及全身藥物分布的背後機制仍需更多探討，具體使用何種基因藥物能夠與本研究相結合尚需更多研究與實驗。

The structural foundation of the blood-brain barrier (BBB) is composed of tightly connected capillary endothelial cells, which effectively prevent the diffusion of molecules that could alter normal brain function. It plays a crucial protective role in maintaining a highly precise brain microenvironment, supporting neuronal activity. However, this also limits the effectiveness of delivering therapeutic nanoparticles for the treatment of central nervous system diseases. Focused Ultrasound (FUS) combined with Microbubbles (MBs) to open the blood-brain barrier (BBB) represents a novel method. This technique is non-invasive, has minimal side effects, and can temporarily open the BBB. It is considered an ideal tool for future gene or drug delivery to the brain. However, in the past, there were two methods of microbubble infusion: Intra-Venous (IV) and Intra-artery (IA). Current research seems to have not discussed in detail the comprehensive discussion of the opening efficacy and systemic drug distribution of these two microbubble infusion methods. In this study, We used FUS open BBB and investigated the differences between intravenous IV and intra-arterial IA drug infusion routes using a small animal model. Evans blue(EB) dye was used as a therapeutic surrogate delivered via IV or IA routes. The extent of BBB opening was evaluated using EB permeability and immunofluorescence staining. Compared to using microbubbles (MBs) alone, the combination of 0.42 MI FUS irradiation and The combination of FUS stimulation and a concentration of 2.8 × 10^8 MBs/kg of MBs significantly increased the EB concentration delivered through IV increased(p = 0.00318) or IA routes(p = 0.0006). Notably, at the same MBs concentration, the IA group’s EB permeability was higher than that of the IV group(p = 0.0276). Increasing the MBs concentration to 1.12×10^9 MBs/kg under FUS further enhanced the EB permeability for both IV(p = 0.0023) and IA groups(p = 0.0008), The IA experimental group achieved the highest EB penetration efficiency. Different FUS stimulation times were also explored. Next, immunofluorescence examination was performed to quantify the brightness of IgG, a marker of BBB permeability. In the substantia nigra (SN) brain region, it was found that under the condition of high concentration of microbubbles, the degree of BBB opening in group IA was better than that in group IV. The safety of the IV and IA experimental groups was also evaluated. Under the same experimental conditions, the bleeding of brain tissue in each experimental group of IA was significantly less than that of the IV experimental group. And IA infusion also has lower performance in systemic drug distribution than IV infusion. The IA experimental group achieved the highest EB penetration efficiency. Additionally, different FUS stimulation times were explored. Immunofluorescence examination was conducted to quantify the brightness of the BBB permeability marker IgG. In the substantia nigra (SN) brain region, under high-concentration microbubble conditions, the IA group’s BBB opening degree was superior to that of the IV group. Finally, the safety of the IV and IA experimental groups was assessed, and under the same experimental conditions, the IA groups exhibited significantly less brain tissue hemorrhage than the IV groups. In conclusion, we demonstrated that drug infusion using IA in conjunction with FUS open BBB enhances targeted delivery to the SN brain region more effectively than IV infusion. The choice of drug infusion route affects the efficiency of BBB opening with FUS, which may be crucial for optimizing gene/drug delivery in the treatment of neurological diseases. However, how the drug infusion route affects the mechanism behind FUS opening of the BBB and systemic drug distribution still needs more exploration, and the specific genetic drugs used in conjunction with this study require more research and experiments.