Investigation of Nanoparticle Accumulation in Tumor Tissues Enhanced by Focused Ultrasound with Microbubbles

本研究目標，是以「非侵入式療法」為立基，利用「超音波」與「微氣泡」，搭配奈米顆粒之使用，促使微血管產生破裂及組織微環境變化，以達「區域性釋放」之效果，強化藥物有效地進入腫瘤組織並增加細胞對藥物之吸收，來大幅提昇藥物之治療效果，及降低其對人體其他正常組織之副作用。 本研究是利用聚焦型超音波及微氣泡來強化奈米顆粒釋放至腫瘤組織累積之定量與定性及最佳參數做深入研究探討。將皮下荷腫瘤Balb/c 小白鼠利用尾靜脈先注射微氣泡(SonoVue®)後，立即施打超音波(操作頻率為1-MHz；峰值聲壓1.2-MPa)，之後經由尾靜脈分別注射4種不同大小之脂質披覆量子點奈米顆粒(30到180 nm)，施打完超音波後約24小時，取下腫瘤組織，隨後透過石墨爐式原子吸收光譜儀、光激發螢光頻譜，和倍頻分子影像顯微鏡對控制組與實驗組分別進行脂質披覆量子點奈米顆粒的定量與定性分析。隨後進一步利用免疫轉印法來分析血管破裂後表現於組織內的生化標誌─P-selectin蛋白質。 本研究亦探討：微氣泡之劑量、超音波震盪時間、治療程序對奈米顆粒在腫瘤組織累積量的影響及奈米顆粒在腫瘤組織中累積隨時間變化，另外亦探討治療後之腫瘤組織再次施打超音波來強化奈米顆粒 之累積量。 由實驗結果可知，聚焦型超音波結合30 uL/kg劑量的微氣泡，能提升腫瘤組織內之奈米顆粒累積，其30、80、130與180 nm奈米顆粒經分析後，在腫瘤組織裡的含量分別為4.47、2.27、0.99與0.82 (ug Cd)/(g tumor); 而不施用微氣泡的對照組，其累積量分別只有1.12、0.75、 0.26與 0.34 (ug Cd)/(g tumor)。在相同的條件下，越小顆粒的奈米粒子在組織內有較高的累積量。再者，免疫轉印法進一步驗證了聚焦型超音波能引起微氣泡震盪/破壞，致使血管壁破裂，使得血管滲透性增加，提升奈米顆粒在腫瘤組織裡的累積。研究結果顯示聚焦型超音波結合微氣泡是一有前瞻性的方法，能促使奈米顆粒有效地進入腫瘤組織內，施打超音波時間之長短與微氣泡之劑量是強化奈米顆粒進入腫瘤組織的最重要因素。Ultrasound-enhanced drug delivery system is a promising technique for noninvasive cancer treatment. Ultrasound-mediated microbubble destruction may enhance the release of nanoparticles from vasculature to tumor tissues. In this study, we used four different sizes of lipid-coated CdSe quantum dot (LQD) nanoparticles ranging from 30 to 180 nm, 1.0-MHz pulsed focused ultrasound (FUS) with a peak acoustic pressure of 1.2-MPa, and an ultrasound contrast agent (UCA; SonoVue®) at a dose of 30 uL/kg to investigate any enhancement of targeted delivery. Tumor-bearing male Balb/c mice were first injected with UCA intravenously, were then sonicated at the tumors with FUS, and were finally injected with 50 uL of the LQD solution after the sonication. The mice were sacrificed about 24 hr after the sonication, and then we quantitatively and qualitatively evaluated the deposition of LQDs in the tumors by using graphite furnace atomic absorption spectrometry (GF-AAS), photoluminescence spectrometry (PL), and harmonic generation microscopy (HGM). Further, immunoblotting analysis served to identify the biochemical markers reflecting the vascular rupture. The experimental results show that the amount of LQDs deposited in tumor tissues was greater in cases of FUS/UCA application, especially for smaller LQDs, being 4.47, 2.27, 0.99, and 0.82 (ug Cd)/(g tumor) for 30, 80, 130, and 180 nm of LQDs, respectively; compared to 1.12, 0.75, 0.26, and 0.34 (ug Cd)/(g tumor) in absence of FUS/UCA. The immunoblotting analysis further indicates that FUS-induced UCA oscillation/destruction results in rupture areas in blood vessels increasing the vascular permeability and thus justifying for the higher quantity of nanoparticles deposited in tumors. Furthermore, we studied the effects of the injected UCA dose (0-300 uL/kg), FUS sonication duration (0-300 sec), and treatment-procedure sequence on the accumulation of nanoparticles in the tumors 24 hr after the treatment, and the time response of the accumulation (0.5-24 hr). After the treatment, the mice were sacrificed and perfused, and then the tumor tissues were harvested for quantifying the amount of nanoparticles using graphite furnace atomic absorption spectrometry (GF-AAS). The results showed that pulsed-FUS sonication with UCA can effectively enhance the vascular permeability for LQD nanoparticle delivery into the sonicated tumors. It indicates that this technique is promising for a better nanodrug delivery for tumor chemotherapy