摘要
背景:
超声微泡介导的超声分子显像及药物和基因靶向传输由于微泡受循环中血液轴流所产生剪切力的影响,效率低下,尤其是在动脉中。超声微泡造影剂在血管腔中的轴流阻碍了微泡与血管内皮的结合,而这个缺点可用超声辐射力克服。
目标:
本研究用低能量超声分别通过体外实验观察不同参数的超声辐射力对微泡造影剂的推移聚集作用,以及在体检测辐射力促进靶向微泡寻靶的能力,目的是为超声分子显像及药物的传递筛选适宜的辐射力参数。
方法:
1.不同参数的超声辐射力对微泡造影剂的作用
建立微血管流体模型,通过体视显微镜观察并记录不同的辐照参数对微泡造影剂的推移、聚集现象,研究不同的超声辐射力参数(声压、频率、微泡浓度、超声辐照时间)对流动状态下的微泡造影剂的推移聚集作用。
2.超声辐射力促微泡造影剂在小血管内的靶向黏附作用
静电吸附法制备携抗ICAM-1抗体的靶向微泡造影剂,并对其进行评价,昆明小鼠阴囊内注射TNF-α构建小鼠提睾肌微血管炎症模型,并随机的分为三组进行实验:①靶向微泡+0KPa辐射力组(对照);②靶向微泡+54.2KPa辐射力组;③靶向微泡+79.3KPa辐射力组,每组5个只。通过激光共聚焦显微镜观察小鼠提睾肌微血管内绿色荧光的面积,以此验证超声辐射力促ICAM-1靶向微泡的在体寻靶能力.
3.超声辐射力促微泡造影剂在大血管内的靶向黏附
亲和素桥接法制备携CD34抗体的靶向微泡造影剂,并分析鉴定。为了进一步证实辐射力在大血管中的作用,本实验以正常大鼠腹主动脉为观察对象,9只SD大鼠随机的分为三组:①靶向微泡+0KPa辐射力组(对照组);②靶向微泡+54.2KPa辐射力组;③靶向微泡+79.3KPa辐射力组。经大鼠尾静脉团注携CD34抗体靶向造影剂的同时进行超声辐照。
扫描电镜观察并记录大鼠腹主动脉后壁内皮表面粘附的微泡情况,利用视觉模糊评分法量化微泡粘附数量,并进行统计学分析。
结果:
1.离体实验
当声强等参数一定时,频率为2.0 MHz的超声波对微泡的推移作用大于1.0 MHz和0.5 MHz,且聚集作用在2.0 MHz时最明显;低声压超声对微泡无明显破坏作用,但使其流速减慢;微泡浓度为7×10~7个/ml和7×10~5个/ml时超声辐射力对微泡有明显推移聚集作用,但微泡浓度高达7×109个/ml时,超声辐射力对微泡的作用不明显;延长辐照时间对微泡的推移和聚集作用无明显影响。
2.小鼠提睾肌微血管在体实验
成功制备携抗ICAM-1抗体的靶向微泡造影剂,浓度为10~7 /ml,粒径范围为1-10μm,90%在3.25μm以下,抗体携带率75%左右。对照组提睾肌微血管内平均荧光为78.0±35.8μm~2,累积光密度为2342.7±1053.1。54.2KPa辐射力组和73.9KPa辐射力组提睾肌微血管内平均荧光面积为422.8±91.3μm~2和1522.0±464.2μm~2,累积光密度为9618.9±2522.0和42123.1±20001.4。单因素方差分析结果显示三组组间比较有明显统计学差异,73.9KPa辐射力组的荧光面积约为对照组的17.5倍,累积光密度约为对照组20倍的。
3.大鼠腹主动脉在体实验
成功制备携CD34抗体的靶向微泡造影剂,浓度为10~7 /ml,粒径范围为1-8μm,90%在2.96μm以下,抗体携带率为70%左右。对照组平均视觉评分为:0.467±0.507分;54.2KPa辐射力组平均视觉评分为1.067±0.785分;73.9KPa辐射力组血管内皮表面可见大量成团的微泡附着,平均视觉评分为3.367±1.189分,单因素方差分析结果显示三组组间比较有明显的统计学差异。
结论:
1.不同参数的超声辐射力对微泡造影剂的推移和聚集作用不同,可望通过调整辐射力参数来最大程度上提高超声微泡造影剂在血液循环中的靶向粘附作用。2.超声辐射力能提高了靶向微泡在微血管内对血管壁靶点的黏附,尤其是当辐射力声压为73.9KPa时靶向粘附率显著提高。3.超声辐射力更能在大血管中发挥其优势对抗高速血流所产生的剪切力,促进微泡的靶向粘附,当辐射力声压为73.9KPa时血管内皮表面能观察到大量微泡的聚集粘附。
Background:
The effectiveness of ultrasound (US) microbubble-mediated molecular imaging and drug delivery has significantly affected by the axial laminar flow of vessel, especially the arteries. Microbubble (MB) UCA exhibits a lateral migration toward the vessel axis in laminar flow, preventing microbubble contact with the endothelium. Recently, it was also found that this limitation could be overcome by using a US radiation force (USRF).
Objective:
In this study, we tried to investigate the abilities of USRF to push microbubble away from central flow and increasing targeted adhesion of circulating targeted microbubble to vessel wall by using different low-amplitude ultrasound radiation forces both in vitro and in vivo. The purpose of this study was to find a set of adaptive USRF parameters, which could be used in molecular imaging and drug delivery.
Methods:
1. Effect of ultrasound radiation force on microbubble contrast agents with different exposure parameters
A capillary flow mimic model was set up for observation and analysis of microbubble displacement and aggregation under stereomicroscope. Investigate the effect of microbubble (MB) contrast agents impacted by different USRF parameters such as acoustic pressure, frequency, duration of exposure, and microbubble concentration under flowing condition.
2. USRF promote the targeted MB adhesion in microvasculature
Prepared targeted microbubble contrast agent which take anti-ICAM-1 monoclonal antibody by means of electrostatic adsorption and evaluated its Character. Construction of the cremaster muscle inflammation model by Scrotum injection TNF-a. Fifteen KM mice were randomized into three groups:①only targeted MB group(control group);②targeted MB + 54.2KPa USRF group;③targeted MB + 73.9 KPa USRF group. The UCA adhesion was observed by laser scanning confocal microscopy and determined by green fluorescence area and integrated optical density(IOD) in cremaster microvasculatures.
3. USRF promote the targeted MB adhesion in abdominal artery
Prepared targeted microbubble contrast agents which take anti-CD34 monoclonal antibody by means of biotin-streptavidin chemistry and evaluated its Character. For examine the ability of USRF promoting UCA retention in great blood vessel. Nine SD rats were divided in to three groups:①only targeted MB group(control group);②targeted MB + 54.2KPa USRF group;③targeted MB + 73.9KPa USRF group. A bolus of 7×10~7 UCA was injected through tail vein and aorta were insonated immediately after UCA injection according to the grouping. The number of retained UCA was count under electronic scanning microscope. we estimated the condition of UCA adhesion in visual analog scale.
Results:
1. In vitro study
The displacement and aggregation of microbubbles occurred significantly at the frequency of 2.0 MHz than 1.0 MHz and 0.5 MHz. Under low acoustic pressure, microbubbles were not visually disrupted but the flow slowed down. MB aggregation and deflection in the tube happened at the MB concentration of 7×10~7/ml but it was not when the concentration rose to 7×10~9/ml because of the high viscosity. The ultrasound exposure time could not affect significantly in displacement and MB aggregation.
2. In vivo study on mouse cremaster microvasculature
Microbubbles carried anti-ICAM-1 monoclonal antibody has been successfully prepared. The diameter of ICAM-1 MB was between 1 to 10μm, with 90% below 3.25μm and the Concentration of it was 107 /ml. The antibody carrying rate of MB was about 75%. The mean fluorescence area in microvascular of control group was 78.0±35.8μm~2, the IOD was 2342.7±1053.1. However, The mean fluorescence area in 52.4KPa and 73.9KPa USRF group were 422.8±91.3μm~2 and 1522.0±464.2μm~2, the IOD were 9618.9±2522.0 and 42123.1±20001.4. There were significant difference between 73.9KPa USRF group and control group using spss13.0 one-way ANOVA test for statistical analysis. The area of green fluorescence are approximately seventeen-fold increase in 73.9KPa group relative to that in control group, and the IOD was twenty-fold in 73.9KPa group compared to the control group.
3. In vivo study on rat abdominal artery
Microbubbles carried anti-CD34 monoclonal antibody has been prepared successfully. The diameter of UCA was between 1 and 8μm, with 90% below 2.96μm and the Concentration of 107 /ml. The antibody carrying rate of MB was about 70%. The mean visual score of control group was 0.467±0.507, and in 52.4KPa USRF group was 1.067±0.785. Contrary to control group, There were a large number of microbubbles attached to the vascular endothelial in 73.9KPa USRF group. Microbubbles were piled layer upon layer and have a tendency of aggregation.
Conclusion:
1. Microbubble contrast agents could be manipulated under some ultrasound parameters. It is expected to physically modulated in blood vessels and helped in targeted adhesions for many therapeutic purposes.
2. USRF can promote the targeted MB adhesion in microvasculatures and the targeting enhanced significantly at a higher acoustic pressure of 79.3 KPa.
3. USRF can be more effective in the large blood vessels to against the shear force generated by axial flow. Substantial targeted retention was observed in the absence of acoustic treatment at a higher acoustic pressure of 79.3 KPa.
引文
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1. Huang SL, Macdonal RC. Acoustically active liposome for drug encapsulation and ultrasound-triggered release. Biochemi Biophys Act. 2004,1665:134-141
2. Behm CZ, Lindner JR. Cellular and molecular imaging with targeted contrast ultrasound. Ultrasound Q. 2006, 22:67-72.
3. Wheatley MA, Lathia JD, Oum KL. Polymeric ultrasound contrast agents targeted to integrins: importance of process methods and surface density of ligands. Biomacromolecules. 2007,8:516-522.
4.杨钰楠,高云华,谭开彬,等.超声介导携RGDS靶向超声造影剂对体外血栓的助溶研究.中华超声影像学杂志.2006, 15:646-626.
5. Liu Y, Miyoshi H, Nakamura M. Encapsulated ultrasound microbubbles: therapeutic application in drug/gene delivery. J Control Release. 2006,114:89-99.
6. Chen S, Shohet RV, Bekeredjian R, et al. Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by ultrasound-targeted microbubble destruction. J Am Coll Cardiol. 2003,42:301-308.
7. Rahim A, Taylor SL, Bush NL, et al. Physical parameter affecting ultrasound/microbubble-mediated gene delivery efficiency in vitro. Ultrasound Med Biol. 2006, 32:1269-1279.
8. Liang H-D, Blomley MJK. The role of ultrasound in molecular imaging. Br J Radiology 2003;76:140–150.
9. Lindner JR. Microbubbles in medical imaging: Current applications and future directions. Nat Rev Drug Discov 2004;3:527–532.
10. Keller MW, Segal SS, Kaul S, Duling BR. The behavior of sonicated albumin microbubbles within the microcirculation: A basis for their use during myocardial contrast echocardiography. Circ Res 1989; 65:458–467.
11. Leong-Poi H, Christiansen J, Klibanov AL, et al. Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted toαⅤ-integrins. Circulation. 2003,107:445-460.
12. Zheng H, Dayton PA,caskey C, et al. Ultrasound-driven microbubble oscillation and translation within small phantom vessels. Ultrasound Med Biol.2007,33(12) :1978-1987.
13. Shortencarier MJ, Dayton PA, Bloch SH, et al. A method for radiation-force localized drug delivery using gas-filled lipospheres.IEEE Trans Ferroelectr Freq Control 2004,51(7):822-831.
14. Dayton PA, Klibanov PA, Brandenburger KE, et al. Acoustic radiation force in vivo: a mechanism to assist targeting of microbubbles. Ultrasound in Med. Biol. 1999,25(8):1195-1120.
15. Joshua J. Rychak, Alexander L. Klibanov,et al. Enhanced targeting of ultrasound contrast agent using acoustic radiation force. Ultrasound in Med. & Biol. 2007,33:1132–1139.
16. Palanchon P, Tortoli P, Bouakaz A, Versluis M, de Jong N. Optical observations of acoustical radiation force effects on individual air bubbles IEEE Trans. Ultrasound Ferroelec Freq Control 2005;52: 104–110.
17. Zhao S, Borden M, Bloch SH, et al. Radiation-force assisted targeting facilitates ultrasonic molecular imaging. Mol Imaging. 2004,3:135-148.
18. Rychak JJ, Klibanov AL, Hossack JA, et al. Acoustic radiation force enhances targeted delivery of ultrasound contrast microbubbles: in vitro verification. IEEE Trans Ultrason Ferroelectr Freq Control. 2005,52:421-433.
19. Fowlkes JB, Gardner EA, Ivey JA, Carson PL. The role of acoustic radiation force in contrast enhancement techniques using bubblebased ultrasound contrast agents. J Acoust Soc Am 1993;93:2348.
20.刘佳,张萍,刘政等,不同参数的超声辐射力对微泡造影剂的作用。中国医学影像技术2010;26:159-161.