环境因素对脂质造影剂的稳定性和回声效率的影响

Influence of environmental factors on the stability and echogenic efficiency of lipid-coated contrast agent

背景目前,利用超声造影剂微泡进行药物或基因的靶向递送的研究越来越多。将载有药物或基因的造影剂微泡在靶向器官上游血管中注入,在靶向器官定位超声,流经靶向器官的造影剂微泡在超声作用下被破坏,产生空化效应,增加血管壁的通透性,促进药物或基因进入到靶向器官和组织中。以磷脂为材料的超声造影剂微泡在药物或基因的靶向给药方面具有显著优势,如磷脂材料无免疫原性、制备工艺简单、容易联接大分子DNA等。本课题组已研制出磷脂为材料的脂质造影剂微泡,利用其可将反义寡聚核苷酸高效地转染到乳腺癌细胞中。目的理想的超声造影剂微泡不但具有良好的超声图像对比增强效果,更应具有足够的稳定性,以便储存供临床应用。本研究利用自制的脂质造影剂微泡研究环境因素对其稳定性和回声效率的影响,并探讨其成因。方法体外试验参照脂质体稳定性考察方法,考察温度、光照和60Co辐射对本品稳定性的影响,以显微形态、微泡浓度和平均粒径作为考察指标。温度设置:放置于低、中、高3种温度下(即0℃、20℃和40℃)保持10d,考察贮存温度对脂质造影剂微泡的影响。光照设置:在(4500±500)Lx强度下强光照射10d,考察光照对脂质造影剂微泡的影响。辐射条件设置:60Co辐射灭菌是冻干粉针剂常用的灭菌方法,本品经过150万rad的60Co辐射,观察辐射对脂质造影剂微泡的影响。动物体内实验采用实时匹配成像技术(CnTi)研究环境因素对脂质超声造影剂回声效率的影响。5只新西兰雄性兔,通过耳静脉的团注脂质造影剂微泡,观察和记录不同实验条件下肝实质区CnTi成像增强强度和持续时间。采用超声仪内置的时间-强度分析软件进行统计分析。结果体外实验结果表明,各实验组微泡浓度均能达到2×109个/ml,且95%的微泡粒径小于8μm。以20℃或40℃贮存组微泡浓度最高,但是各组条件的显微形态和平均粒径结果相差不明显(表1,图1);动物CnTi成像实验表明,40℃贮存组肝实质区成像增强强度和持续时间最高,其次是20℃贮存组和60Co辐射灭菌组,而强光照射组和0℃贮存组成像增强强度和持续时间最差(表2,图2)。不同的温度实验结果的解释与磷脂的相转变温度(Tc)有关;本品磷脂材料的Tc为42℃～45℃,当环境温度小于本品磷脂的Tc时,磷脂膜为胶晶相结构,排列紧凑而有序,具有较好的弹性(图3)。但是温度降到接近0℃时,磷脂胶晶相结构会变得松脆。而温度超过Tc时,磷脂膜会从胶晶相向液晶相转变,磷脂膜会液化消失。因此尽管其显微形态和平均粒径相差不明显,0℃贮存时因磷脂膜变得松脆而导致其回声效率显著降低,成像增强强度和持续时间最差。贮存于20℃和40℃时,磷脂膜具有较好的弹性,因此成像增强强度和持续时间较好。光照会诱发磷脂的氧化反应,降低脂质微泡膜的弹性。因此强光照射组显示出与0℃贮存实验组相近的结果,即尽管其显微形态和平均粒径变化不大,但是CnTi成像增强强度和持续时间差。60Co辐射对脂质超声造影剂影响较小,显微形态、平均粒径和CnTi成像效果均未见显著影响,说明60Co辐射可以作为本品的灭菌方法。结论本品在避光条件下常温保存,其稳定性较高,并能采用60Co辐射灭菌。

Background Recently,there have been numerous reports on application of insonated microbubbles for targeting or controlling drug/gene release.The microbubbles can be injected upstream of the target region,and then that target region is easily imaged at low intensity by the presence of the bubbles.When the imaging demonstrates that ultrasound is precisely focused on the target tissue,then the ultrasonic intensity can be increased to create collapse cavitation.The collapse events appear to permeabilize the vessel walls and provide pathways for extravasation of drug/gene that is freely floating along with the microbubbles,or that is associated with the microbubble surface.Phospholipids-based microbubbles represented a popular avenue for drug/gene transfer because their distinct advantages include(1)robust manufacture,(2)ease in handling and preparation techniques,(3)ability to deliver large DNA molecules,and(4)low immunogenic response.Previous work in this laboratory has prepared lipid-coated microbubbles capable of transfer antisense oligodeoxyribonucleotide into breast cancer cells.Objective A successful insonated microbubble carrier must not only withstand the static pressure and tension effects of the human vasculature,but also have the stability to resist ambient conditions during storage for it to be used clinically.In this work,experiments were carried out to evaluate possible effects of some environmental factors on the echogenic efficiency of insonated lipid-coated microbubbles in normal rabbit liver parenchyma.Methods 1.Stability in vitro:stability testing were performed to evaluate some important factors affecting lipid-coated contrast agent stability in vitro-three levels of storage temperature,high lighting and 60Co eradiation sterilization.Morphological characteristics,concentration and the diameter of microbubbles were considered as indices.Temperature tests set:low temperature(0℃),high temperature(40℃)and normal temperature(20℃).High lighting tests set:4500±500 Lx.Samples of temperature and high lighting tests were collected at 10th day.60Co eradiation sterilization was prepared by eradiating agents with 60Co at 1.5 million rad,which ensured the agents sterilization.2.Echogenic efficiency in vivo:5 New Zealand white male rabbits were given contrast agent by bolus injections.Contrast tuned imaging(CnTi)technique was used to assess the relationship of different acoustic pressures and enhancement effects in normal rabbit liver parenchyma.Changes in the video intensity of liver parenchyma were analyzed by the built-in software.Results As the results obtained from the stabili in vitro showed,the concentration of lipid-coated microbubbles reached above 2×109 microbubbles/ml,with 95% of the microbubbles below 8 μm in diameter.Storage at 20℃ or 40℃ resulted in higher concentration of microbubbles than other conditions(storage at 0℃,high lighting and 60Co eradiation sterilization),but we found no significant difference among the micromorphology and mean diameter(P>0.05)(Tab 1,Fig 1).In CnTi study,peak intensity and duration of CnTi enhancement in the liver parenchyma were observed at the condition of storage at 40℃,followed with storage at 20℃ and 60Co eradiation sterilization.High lighting and storage at 0℃ resulted in low contrast intensity and short duration of CnTi enhancement(Tab 2,Fig 2).The reasons of different results under temperature tests were related to phase transition temperature(Tc).The lipid membrane of lipid-coated microbubbles were formed by hydrogenated phosphatidylcholine(HPC),whose Tc was 42-45℃.Below Tc,the membrane structure of lipid-coated microbubbles was formed orderly and compactly(Fig 3).However,the fragility of the lipid membrane was increased when the temperature of storage decreased to 0℃.When environmental temperature raised beyond Tc,lipid membrane was liquified and membrane structure was lost.Therefore,the lipid membrane at 0℃ lost adequate elasticity and resulted in low contrast intensity and short duration of CnTi enhancement,though showed similar morphological characteristics and mean diameter as other tests.In contrast,the lipid membrane at 20℃ and 40℃ maintained adequate elasticity and resulted in high contrast efficiency.It is well known that the light induces many oxidative reactions.In our experiment,high lighting reduces the elasticity of the membrane of microbubbles by inducing the oxidation of HPC.Therefore,high lighting test has the same in vitro and in vivo results as that of storage at 0℃.From the experiment,60Co eradiation sterilization has little effect on lipid-coated microbubbles stability in vitro and in vivo,which provides an effective method of sterilization for lipid-coated microbubbles.Conclusions As demonstrated by these experiments,lipid-coated contrast agent should be kept at room temperature in the dark during storage.60Co eradiation could be used as a sterilization method in preparation.