RGD-L-TRAIL@磁性微气泡的构建及其用于结肠癌分子影像合并实时治疗的方法研究

发布时间：2018-06-25 02:41

本文选题：微气泡 + 磁性纳米颗粒　；参考：《东南大学》2017年博士论文

【摘要】：近年来,随着微纳载体材料的不断发展,磁性纳米颗粒和微气泡已分别发展成为有效的医学影像造影剂和药物输送载体系统。融合两者的优点制备的携带磁性纳米颗粒的磁性微气泡载体材料,由于兼具优良的磁学特性和声学特性在生物医学应用领域得到了较广泛的关注和研究。而将磁性微气泡与药物、抗体、多肽或多糖结合起来,形成集早期诊断与精确靶向治疗于一体的多模式、多功能造影剂,更是目前生物医疗造影材料的一个发展趋势。本论文将超顺磁性APTS/γ-Fe203纳米颗粒在聚合物微气泡表面进行了可控组装并深入探讨了组装机理,在对组装后的磁性微气泡进行膜壳性质、多模态成像性能等方面深入研究的基础上,将一种兼具靶向与治疗功能的RGD-L-TRAIL蛋白与之结合,构建了RGD-L-TRAIL磁性微气泡分子探针,此探针既能靶向于肿瘤新生血管并诱导肿瘤细胞凋亡,在病灶部位富集之后,又可以利用微气泡的声学特性进行超声显影,利用γ-Fe2O3纳米颗粒的磁学特性进行磁共振成像,从而达到了诊疗一体化的效果。主要在如下几个方面进行了研究:1.对聚合物微气泡在稳定性及粒径方面进行了优化,通过端基羧基化修饰有效提高了微气泡的分散稳定性及耦联其它活性物质的能力,通过不断优化实验条件,使微气泡粒径更加均匀,在保持超声成像增强能力的同时,更适于体内应用,为后续工作打下了良好的基础。2.通过"表面装载"的方式在聚合物微气泡上组装了不同浓度梯度的APTS/γ-Fe203超顺磁性纳米颗粒,对组装机理及微气泡膜壳结构的变化进行了深入探讨,对磁性微气泡的磁性、稳定性、磁共振/超声成像效果进行了考察,结果表明这种表面装载磁性纳米颗粒的磁性微气泡具有"高装载量","高磁性",MRI/US显影效果的"可调控性"及潜在的耦联其它活性物质的特点,显示出了良好的生物医学应用潜力。3.在上述磁性微气泡表面,偶联了靶向肿瘤新生血管和诱导肿瘤细胞凋亡的RGD-L-TRAIL蛋白,形成了磁性微气泡分子探针。对此探针进行了粒径、磁滞回线、显微形貌等结构表征及稳定性考察;对磁性微气泡表面偶联的RGD-L-TRAIL蛋白进行了定量分析,优化出了使磁性微气泡分子探针兼具灵敏性和稳定性的"最合适"蛋白偶联量。4.采用频率为3.5MHz的临床超声诊断仪,对优化后的磁性微气泡分子探针进行了体外超声成像实验,结果表明其可以有效增强超声成像效果,维持时间大约在5分钟左右。同时,采用7T磁共振成像仪,对磁性微气泡分子探针进行了体外磁共振成像研究,结果表明其可以有效降低磁共振成像时的T2信号。从而证实RGD-L-TRAIL磁性微气泡分子探针具有超声/磁共振双模态造影的功能效果。5.在体外,分别检测了未偶联RGD-L-TRAIL蛋白的磁性微气泡,偶联蛋白的磁性微气泡及相同浓度的纯RGD-L-TRAIL蛋白诱导结肠癌细胞COLO-205凋亡的情况,结果显示磁性微气泡分子探针可以很好地诱导COLO-205细胞凋亡,而相同剂量的未偶联RGD-L-TRAIL的磁性微气泡不具有诱导凋亡的能力。从而可以证实在与磁性微气泡偶联后,RGD-L-TRAIL蛋白保留了诱导细胞凋亡的活性,且凋亡具有蛋白剂量依赖性。6.建立了裸鼠结肠癌肿瘤模型,分别以瘤内及尾静脉两种注射方式,将RGD-L-TRAIL磁性微气泡分子探针注入裸鼠体内,用小动物超声成像系统进行了肿瘤部位的超声成像实验,并与未偶联RGD-L-TRAIL蛋白的磁性微气泡进行比较,结果发现在两种注射方式下,RGD-L-TRAIL磁性微气泡分子探针均会使肿瘤部位超声图像具有更高的灰度值,且维持更长的灰度保持时间。同时,采用7T磁共振成像仪进行了肿瘤部位的磁共振成像实验,结果表明,无论用何种注射方式,RGD-L-TRAIL磁性微气泡分子探针引起的T2信号强度变化均大于无蛋白偶联的磁性微气泡。以上结果证实了磁性微气泡分子探针在体内可以靶向肿瘤细胞,更有效地同时增强超声/磁共振双模态成像效果。此外,还通过对肿瘤部位组织切片中Fe及RGD-L-TRAIL蛋白的检测,进一步验证了磁性纳米颗粒、微气泡和RGD-L-TRAIL蛋白的有效耦联,及磁性微气泡分子探针的靶向性。7.最后,考察了 RGD-L-TRAIL磁性微气泡分子探针体内抑制肿瘤的效果。结果表明,尾静脉注射RGD-L-TRAIL蛋白及RGD-L-TRAIL磁性微气泡分子探针均可抑制结肠癌移植瘤的生长,肿瘤抑制率分别为44.6%和47.5%,而未偶联蛋白的磁性微气泡无法有效抑制肿瘤生长,这一结果写TDNEL法检测各组动物肿瘤细胞调亡情况相一致。
[Abstract]:In recent years, with the continuous development of micro nano carrier materials, magnetic nanoparticles and microbubbles have developed into effective medical imaging agents and drug delivery carriers, respectively. The magnetic microbubble carrier material, which combines the advantages of the two, has excellent magnetic and acoustic properties. The magnetic microbubbles are combined with drugs, antibodies, peptides or polysaccharides to form a multi mode, multi-functional contrast agent, which is a combination of early diagnosis and precise target therapy, and is a development trend of biological medical contrast materials. This paper will be a superparamagnetic APTS/ gamma -Fe in this paper. 203 nanoscale particles were assembled on the surface of polymer microbubbles and discussed the mechanism of assembly. On the basis of a thorough study of the properties of the magnetic microbubbles after the assembly and the multi-modal imaging performance, a kind of RGD-L-TRAIL protein with both target and therapeutic function was combined to construct a RGD-L-TRAIL magnetic microsphere. The probe can target the neovascularization and induce the apoptosis of tumor cells. After enrichment of the tumor, the probe can be used to develop the ultrasonic imaging using the acoustic characteristics of microbubbles. The magnetic resonance imaging of gamma -Fe2O3 nanoparticles is used to achieve the effect of integration of diagnosis and treatment. Research is carried out in the following aspects: 1. the stability and particle size of polymer microbubbles are optimized. Through the carboxylation of the end group, the dispersion stability of microbubbles and the ability of coupling other active substances are effectively improved. By optimizing the experimental conditions, the particle size of microbubbles is more uniform, while the enhancement ability of ultrasonic imaging is maintained. It is more suitable for the application in the body and lays a good foundation for the follow-up work..2. has assembled the APTS/ gamma -Fe203 superparamagnetic nanoparticles with different concentration gradient on the polymer microbubbles through the "surface loading" way. The assembly mechanism and the change of the microbubble membrane shell structure are deeply investigated. The magnetic, stability and magnetic co magnetic properties of the magnetic microbubbles are discussed. The effect of vibration / ultrasonic imaging was investigated. The results showed that the magnetic microbubbles loaded with magnetic nanoparticles had "high loading", "high magnetic", "controllable" effect of MRI/US development and potential coupling of other active substances, showing good biomedical application potential.3. on the surface of the above magnetic microbubbles. The RGD-L-TRAIL protein was coupled to the neovascularization of the tumor and the apoptosis of the tumor cells, and the magnetic microbubble molecular probe was formed. The probe was characterized by particle size, hysteresis loop, micromorphology and other structural characterization and stability. The quantitative analysis of the RGD-L-TRAIL protein coupled with the surface of magnetic microbubbles was carried out to optimize the magnetic properties. The microbubble molecular probe with the "most suitable" protein coupling quantity of sensitivity and stability.4. uses a clinical ultrasonic diagnostic instrument with frequency of 3.5MHz. The ultrasonic imaging experiments of the optimized magnetic microbubble molecular probe have been carried out. The results show that it can effectively enhance the effect of ultrasound imaging, and the maintenance time is about 5 minutes. 7T magnetic resonance imaging (MRI) was used to study magnetic resonance imaging (MRI) of magnetic microbubbles in vitro. The results show that it can effectively reduce the T2 signal in magnetic resonance imaging. Thus, it is proved that the RGD-L-TRAIL magnetic microbubble molecular probe has the functional effect of ultrasonic / MRI dual mode contrast and.5. in vitro, respectively, to detect uncoupled RGD-L-. The magnetic microbubbles of TRAIL protein, the magnetic microbubbles of the coupling protein and the pure RGD-L-TRAIL protein of the same concentration induced the apoptosis of COLO-205 in colon cancer cells. The results showed that the magnetic microbubble molecular probe could induce the apoptosis of COLO-205 cells well, and the same dose of uneven RGD-L-TRAIL magnetic microbubbles did not have induced withering. It was proved that after coupling with magnetic microbubbles, RGD-L-TRAIL protein retained the activity of inducing apoptosis, and apoptosis had a protein dose dependent.6. to establish the tumor model of nude mice with colon cancer, and the RGD-L-TRAIL microbubble molecular probes were injected into nude mice with two intratumoral and caudal vein injection methods respectively. The ultrasonic imaging experiments of tumor sites were carried out with small animal ultrasound imaging system and compared with the magnetic microbubbles without RGD-L-TRAIL protein. The results showed that under the two injections, the RGD-L-TRAIL magnetic microbubble probe would have a higher gray value in the tumor location and maintain a longer gray level. At the same time, the magnetic resonance imaging experiment of the tumor site was carried out by 7T magnetic resonance imaging instrument. The results showed that the intensity of T2 signal caused by RGD-L-TRAIL magnetic microbubble molecular probe was greater than that of non protein coupling magnetic microbubbles. The above results confirmed that the magnetic microbubble molecular probe was in vivo In addition, the effective coupling of magnetic nanoparticles, microbubbles and RGD-L-TRAIL proteins, and the target.7. of magnetic microbubble molecular probes are further verified by the detection of both Fe and RGD-L-TRAIL protein in the tissue section of the tumor. The effects of RGD-L-TRAIL magnetic microbubbles on tumor inhibition were investigated. The results showed that the growth of colon cancer transplanted tumor was inhibited by RGD-L-TRAIL protein and RGD-L-TRAIL magnetic microbubble molecular probe injected in the tail vein, and the tumor inhibition rates were 44.6% and 47.5% respectively, while the uncoupled protein magnetic microbubbles could not effectively inhibit the tumor. This result was consistent with the TDNEL method in detecting the apoptosis of tumor cells in each group.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.35

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