携带适配体AS1411的液态内核纳米超声造影剂的制备和评估

目的：探讨制备核酸适配体AS411靶向液态内核的纳米超声造影剂的方法，评价该靶向纳米超声造影剂 体外显影能力和寻靶能力。方法：将自合成的膜材料聚乳酸-羟基乙酸共聚物-聚乙二醇(PLGA-PEG-COOH)和全氟辛 溴烷(perfluoroctylbromide，PFOB)，采用乳化溶剂挥发法，制备液态内核的纳米颗粒(nanoparticles，NP)。然后用1- 乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)/N-羟基琥珀酰亚胺(NHS)催化法将AS1411连接到NP表面，制成靶向液态内 核的纳米颗粒(NP-AS1411)。用透射电镜检查NP-AS1411的形态。比较NP-AS1411和NP的大小、表面电荷、包封率、 生物相容性、体外显影能力和稳定性。用凝胶电泳检查AS1411是否连于NP表面。用荧光显微镜和流式细胞仪检测 NP-AS1411的靶向能力。结果：NP-AS1411在电镜下呈壳-核结构，直径为(245.4±16.5) nm，大于NP的直径(P=0.05)。 NP-AS1411和NP在表面电荷和包封率上差异无统计学意义(P>0.05)。高浓度(25.0 mg/mL)NP-AS1411与MCF-7细胞孵育 24 h后，细胞的活力(89%)和对照组相比明显下降(P=0.04)。NP-AS1411体外相对灰阶值为86.1±6.7，明显高于脱气去 离子水(P<0.05)，与和NP的体外相对灰阶值相当(P>0.05)。常温保存24 h后，NP-AS1411的相对灰阶值为80.1±9.2，与 24 h前相比差异无统计学意义(P>0.05)。NP-AS1411的大小在放置24 h后也无明显变化(P>0.05)。凝胶电泳证实当NP和 AS1411的摩尔比为40:1时，连接到NP的AS1411最多。MCF-7细胞分别与载有荧光香豆素6的NP-AS1411，NP孵育后， NP-AS1411在荧光显微镜下能够产生更强的绿色荧光。流式细胞仪证实NP和NP-AS1411与MCF-7细胞均有结合，但是 NP-AS1411和MCF-7细胞结合后产生的荧光更强烈。结论：采用乳化溶剂挥发法和EDC/NHS催化法可以成功制备适配 体靶向液态内核纳米超声造影剂。该靶向液态内核纳米超声造影剂的体外显影能力好，也同时具有良好的稳定性和 特异性。

Development and evaluation of ultrasound contrast agents with AS1411-conjugated nanoparticles with liquid core

Objective: To prepare AS1411 targeted nano-ultrasonic contrast agent with liquid core, and to evaluate its ability for ultrasonic contrast enhancement and targeting MCF-7 cell in vitro. Methods: The modified solvent evaporation, self-synthesized membrane material and perfluorobrominane (PFOB) was used to form nano-ultrasonic contrast agent with PFOB core (nanoparticles, NP); then N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) catalysis was used to connect AS1411 to the surface of NP to prepare NP-AS1411. The transmission electron microscopy was chosen to check the morphology of NP-AS1411. The size, surface charge, encapsulation efficiency, biocompatibility, the contrast grey value and the stability of NP-AS1411 and NP were compared. Whether AS411 was attached to the surface of NP was checked by gel electrophoresis. Fluorescence microscopy and flow cytometry were performed to examine the targeting ability of AS1411. Results: NP-AS1411 was a shell-nuclear structure under the electron microscope. Its size was at (245.4±16.5) nm, which was larger than that of NP (P=0.05). There was no significant difference in surface charge and encapsulation efficiency between NP-AS1411 and NP (P>0.05). In the MTT experiment, the cell viability decreased significantly at high concentration of NP-AS411 (25 mg/mL) after incubation for 24 h compared with the control group (0 mg/mL ) (P<0.05). The contrast gray value of NP-AS1411 was at 86.1+ 6.7, which was significantly higher than that of deionized water (P<0.05), and equivalent to that of NP (P>0.05). The contrast grey value of AS1411-NP was 80.1±9.2 after keeping at room temperature for 24 h, which showed no obviously change comparing with that before the treatment (P>0.05). The size of NP-AS1411didn’t change too (P>0.05). The results of gel electrophoresis demonstrated that the AS1411 connecting to the surface of NP was the most when the molar ratio of NP:AS1411 was at 40:1. Flow cytometry analysis confirmed that NP and NP-AS1411 were combined with MCF-7 cells separately but the fluorescence produced by the combination of NP-AS1411 and MCF-7 was more intense. Conclusion: The modified solvent evaporation and EDC/NHS catalysis could successfully prepare ultrasound contrast agents with aptamer-conjugated nanoparticles with liquid core. The targeted ultrasonic contrast agents with liquid core possess good ultrasonic contrast enhancement ability in vitro, stability and specificity as well.