治疗超声联合耦连载MTX纳米粒微泡促药物跨血脑屏障转运的实验研究

发布时间：2018-05-31 18:12

本文选题：血脑屏障 + 超声　；参考：《第三军医大学》2013年博士论文

【摘要】：背景和目的血脑屏障(Blood brain barrier，BBB)对维持中枢神经系统内环境的稳定具有重要作用，也正是由于其本身结构功能的特点, BBB阻碍了许多治疗中枢神经系统疾病药物的进入，无论是口服还是经血管给药，药物都很难通过BBB从而进入脑组织，无法在脑组织内达到有效治疗浓度而实现其治疗作用，影响治疗效果。研究表明，治疗中枢神经系统疾病药物的疗效主要是取决于血脑屏障对该药物的通透性。直接脑室穿刺给药有创伤性且容易伴发感染，限制了其应用；而对药物进行相关或一定修饰、改变分子量大小和电荷，针对葡萄糖、氨基酸、肽类等通过BBB的特殊载体，因给药效率低，目前还难以广泛应用于临床。因此，如何无创、可逆、靶向地开放BBB，使治疗药物透过血脑屏障，在脑区达到药物有效治疗浓度是目前国内外学者普遍关注的热点之一。超声联合微泡辐照脑组织，已证实可以实现可逆、无创性增强血脑屏障通透性，其开放血脑屏障的机制可能与空化效应有关。目前，应用超声波联合新型造影剂微泡进行药物跨BBB转运也为超声治疗开创了新的平台，超声微泡造影剂可用作药物运载体，将药物包载于造影剂微泡中防止药物在体循环中降解失活，结合超声靶向微泡击破（ultra-sound targetedmicrobubble destroy，UTMD）技术，可以选择性的将药物在目的区域释放。利用该方法，一方面增加了药物的局部作用浓度，另一方面也降低了血液中的药物浓度，从而实现在减少全身性副作用的同时提高药物疗效。 “脂氟显”是我科自行研制并具有独立自主知识产权的新型脂膜超声造影剂，经过前期大量动物实验研究证实，无论是在诊断还是在治疗领域，均取得良好的应用研究结果。但载药量较低也成为我们将应用“脂氟显”于治疗领域的一个障碍，同时回顾目前载药物或载基因超声造影剂的相关研究，超声造影剂作为药物或基因载体均有其不可避免的局限性，一方面，造影剂微泡外壳为较薄的脂膜或蛋白膜，在膜上能携带的药物量及基因量均有限，通过透射电镜对“脂氟显”进行检测也证实其外壳由非常薄的膜构成；另一方面，造影剂微泡中心为惰性气体所占据，这就更减少了造影剂微泡携带药物及基因的能力。因此如何提高超声造影剂微泡的载药量成为目前急待解决的问题。纳米微粒是近年来研究较多的新型药物载体，其载药的有效性及其控制释放药物能力在大量研究中得到了证实，尤其是脂质体载药纳米粒，由双层脂膜中心包裹水性核构成，其脂膜上具有携带亲脂性药物及两亲性药物，而中心部的液性部分亦可携带亲水性药物，因此其携载药物的种类较造影剂微泡更广泛。同时，脂质体作为药物载体能降低药物的给药剂量，减轻药物毒性，提高药物的稳定性。研究表明，载药脂质体能使药物在肿瘤部位聚集达到单纯应用药物的50~100倍。但要达到不同部位靶向释放，则需要不同修饰方法进行脂质体的制备。若脂质体能在超声波作用下实现目的治疗区的靶向释放则可减少制备工艺上的烦琐过程，然而，由于脂质体的核心为液态物质，经注射入血液后并不能与超声波产生协同作用的效果，因此超声亦无法对纳米粒实现实时的监测，对于其是否到达目的治疗区亦无从掌握。基于上述“脂氟显”及载药纳米粒的相关特点及其不足，本研究拟在现有制备“脂氟显”的基础上，于其表面耦连上载药脂质体纳米粒，由于脂质体纳米粒粒径较小，造影剂微泡耦连上纳米粒后，其粒径的总体变化不会发生太大的改变，即超声造影剂微泡在超声波声场中的相关物理特性不会发生改变，这样我们就可以解决造影剂微泡载药量低的缺陷，同时利用超声波能有效击破微泡从而在特定部位释放微泡膜上耦连的载药纳米粒，实现脂质体纳米粒在超声辐照场中靶向释放药物的的效果。聚乙二醇(PEG)是脂质体制备中较常用的修饰材料，脂质体中引入PEG后可有效地降低调理素对脂质体纳米粒的亲和性，减少肝脏巨噬细胞对载药脂质体纳米粒的吞噬作用，从而延长脂质体在血液中的循环时间，同时，无论是脂质体纳米粒还是造影剂微泡在引入PEG后均能为各自的膜构成上提供有效的空间构架，在此基础上仅对PEG进行一定的修饰即可实现脂质体纳米粒与造影剂微泡的耦连，从而实现本研究中制备新型超声造影剂的设想。由于血脑屏障的作用，化疗药物不易渗入脑膜、眼眶等“盲区”，这些部位的残留白血病细胞是造成中枢神经系统白血病(central nervous system leukemia, CNSL)复发的主要根源。目前，颅脑放疗、鞘内注射化疗药物和大剂量甲氨蝶呤（Methotrexate,MTX）化疗是预防CNSL复发的主要措施，但放疗的后期副作用明显，对内分泌、神经系统均有毒性作用；鞘内注射化疗药物具有创伤性，且易引发感染；大剂量甲氨蝶呤(HDMTX)是目前较为有效的CNSL预防方法之一，但亦存在较大的副作用，同时还可以导致远期的精神毒性。为此，如何使MTX在脑组织病灶内达到有效浓度，并降低其毒副作用，是治疗和预防CNSL的关键所在。本研究目的是在前期制备“脂氟显”造影剂的基础上，制备高效载药的载MTX纳米粒，利用生物素-亲和素桥接法，将载药纳米粒与脂质微泡耦连，制得耦连载MTX纳米粒的新型载药造影剂，，并观察其安全性及在超声辐照场中体外释放药物的效果；同时探讨治疗超声联合耦连载MTX纳米粒造影剂促药物跨大鼠血脑屏障转运的能力，并对其机理进行初步探讨，为中枢神经系统疾病治疗探索一种无创、安全的新方法。方法及路线 1.在“脂氟显”制备工艺基础上，制备生物素化“脂氟显”超声造影：将一定比例生物素化磷脂加入原“脂氟显”脂质配方中，适当改变磷脂配比，以库尔特粒度计数仪对制备的造影剂进行粒径分布及浓度进行测定。 2.在“脂氟显”制备工艺基础上，制备生物素化载MTX脂质纳米粒：利用二次冷冻干燥法及机械振荡法制备生物素化载MTX的脂质纳米粒，应用柱层析法实现载药纳米粒与游离药物的分离，应用高效液相色谱法对载药纳米粒进行包封率测定，应用Zetasizer3000对纳米粒进行粒径分布测定，透射电镜进行形态学及粒径分布观察与测量。 3.生物素化“脂氟显”造影剂与载MTX脂质纳米粒的耦连：制备不同含量生物素化的“脂氟显”造影剂及载MTX脂质纳米粒，利用亲和素－生物素连接体系对两者进行耦连，以漂洗－离心法分离未结合的纳米粒，最终得到耦连载药纳米粒的超声造影剂，应用Coulter Counter对耦连载药纳米粒超声造影剂进行粒径测定；利用制备的造影剂对大鼠肝脏进行造影检查，以了解其增强显像效果；应用高效液相色谱法测定耦连载药纳米粒造影剂MTX携带量。 4.耦连载MTX脂质纳米粒微泡体外对肿瘤细胞抑制效果及机制研究：以一定功率治疗超声仪，辐照加入耦连载MTX脂质纳米粒微泡后的肿瘤细胞悬液，应用MTT法及凋亡检测手段测量其对肿瘤细胞的抑制率，并应用扫描电镜观察超声与微泡联合作用后细胞形态变化，以初步了解其作用机制。 5.超声联合微泡开放大鼠血脑屏障的实验研究：应用治疗超声仪联合微泡辐照大鼠颅脑，选择不同参数，包括功率、辐照时间、占空比、微泡用量等，以伊文思蓝(Evans-blue, EB)示踪实验评价大鼠BBB开放情况，筛选出最佳辐照参数，并评价其安全性及可逆性。 6.超声联合载药微泡促MTX跨大鼠血脑屏障转运的实验研究：分为四组：(1)超声+载药微泡组；(2)超声+微泡+药物组；(3)载药微泡组；(4)药物组。利用高效液相色谱法检测各组脑组织MTX浓度，并通过硝酸镧示踪实验对其机制进行初探。 7.体外血脑屏障模型的建立：原代培养BALB/c小鼠脑星形胶质细胞，将小鼠脑微血管内皮细胞(Brainmicrovasular endothelial cell，BMVEC)与星形胶质细胞分别接种于transwell小室多孔滤膜的两面进行共培养，通过跨膜电阻测量，通透性测试，免疫组化ZO-1检测，透射电镜检查评价其形态学及限制通透功能。结果及结论 1.应用冻干技术可以成功制备生物素化超声造影剂，利用二次冻干技术可以实现载甲氨蝶呤脂质纳米粒的制备，该制备方法工艺流程相对简单。 2.葡聚糖凝胶柱层析法可以有效实现载药纳米粒与游离药物的分离。 3．亲和素－生物素连接体系可以实现脂质微泡与载药纳米粒的耦连，此法值得的载药造影剂粒径符合静脉注射要求，有较高载药量，载药量约(4.91±0.51) mg/ml。 4.超声联合耦连载MTX纳米粒微泡能有效抑制肿瘤细胞的增殖，其作用机制与其在细胞表面形成孔洞有关。 5.采用MetronAP-170超声波治疗仪，探头频率1MHz，输出功率2.0W/cm2、占空比20%、辐照时间5min、造影剂剂量0.5ml/kg，可以安全可逆的开放大鼠血脑屏障。 6.采用上述参数，超声联合载药微泡可以明显增加MTX跨大鼠血脑屏障转运，与其余三组比较有显著性差异(P0.01)，其机制可能与造影剂微泡在超声波的作用下产生的空化作用，引起了血脑屏障紧密连接开放有关。 7．脑微血管内皮细胞与星形胶质细胞共培养模型，在形态学，通透性及屏障性方面具备了BBB的基本特征，可用于药物跨血脑屏障转运的研究。
[Abstract]:Background and purpose
Blood brain barrier (BBB) plays an important role in maintaining the stability of the environment in the central nervous system. It is also due to its own structure and function. BBB hinders the entry of many drugs in the central nervous system. It is difficult to enter the brain through BBB, whether oral or via blood vessel. The therapeutic effect of the method is achieved in the brain tissue to achieve its effective therapeutic concentration and effect the therapeutic effect. The study shows that the therapeutic effect of the drugs for the central nervous system disease is mainly dependent on the permeability of the blood brain barrier to the drug. As a special carrier of BBB, such as glucose, amino acids, peptides, and so on, it is still difficult to be widely used in clinic because of the low efficiency of drug delivery.
Therefore, it is one of the hot spots for scholars both at home and abroad to open the BBB without invasive, reversible and targeted, so as to make the therapeutic drugs through the blood brain barrier and to reach the effective treatment concentration in the brain area. The system may be related to the cavitation effect. At present, the use of ultrasound combined with a new contrast agent microbubble to translocate the drug across BBB also creates a new platform for ultrasound treatment. Ultrasound microbubbles can be used as a drug carrier, and the drug is loaded in the contrast agent microbubble to prevent the deactivation of drugs in the body based ring, and UL Tra-sound targetedmicrobubble destroy, UTMD) technology can selectively release drugs in the target area. By this method, the local concentration of drugs is increased on the one hand, and the drug concentration in the blood is reduced on the other, thus reducing systemic side effects and improving the efficacy of the drug.
"Lipo fluorine" is a new type of lipid membrane ultrasound contrast agent developed by our family and has independent intellectual property rights. It has been confirmed by a large number of animal experiments in the earlier period, and it has achieved good results in both diagnosis and treatment. But the low drug loading has also become one of the fields in which we will apply lipid fluorine in the field of treatment. At the same time, a review of the current studies on drug or gene carrier ultrasound contrast agents. Ultrasound contrast agents have their unavoidable limitations as drugs or gene carriers. On the one hand, the microbubble shell of the contrast agent is thinner lipid membrane or protein membrane, and the amount of drugs and the amount of genes that can be carried on the membrane are limited. Transmission electron microscopy is used for "lipid". The detection of fluorine shows that the shell is made up of very thin films; on the other hand, the center of the microbubble is occupied by the inert gas, which reduces the ability of the contrast agent to carry drugs and genes. Therefore, how to improve the drug loading of the contrast agent microbubbles has become an urgent problem to be solved before the eye.
Nanoparticle is a new drug carrier which has been widely studied in recent years. The effectiveness of its drug loading and its ability to control the release of drugs have been confirmed in a large number of studies, especially liposomal drug loaded nanoparticles, consisting of a bilayer lipid membrane core wrapped in water core, with lipid pericyclic drugs and two Pro drugs on the lipid membrane, and the liquid nature of the central part. Some can also carry hydrophilic drugs, so the species carrying drugs are more widely used than contrast agent microbubbles. At the same time, liposomes as drug carriers can reduce the dosage of drugs, reduce drug toxicity and improve the stability of drugs. The study shows that the drug loaded liposomes can gather the drug in the tumor site to reach the 50~100 times of the use of the drug. However, in order to achieve the target release in different parts, the preparation of liposomes is required by different modification methods. If the target release of the target treatment area is realized under the action of the liposome, it can reduce the cumbersome process in the preparation process. However, because the core of the liposome is liquid substance, the injection of the liposome into the blood can not be produced with the ultrasound. Therefore, ultrasound can not monitor the nanoparticles in real time, and it is impossible for them to reach the target treatment area.
Based on the characteristics and shortcomings of the "lipid fluorine" and drug loaded nanoparticles, this study is designed on the basis of the current preparation of "lipid fluorine" and on the surface of the liposome nanoparticles. The overall change of the particle size will not be greatly changed since the nanoparticle size of the liposome is smaller and the contrast agent microbubbles are coupled to the nanoparticles. That is, the physical characteristics of the ultrasound contrast agent microbubbles in the ultrasonic sound field will not change, so that we can solve the defects of the low dose of the microbubbles in the contrast agent. At the same time, we can use the ultrasonic wave to effectively break the microbubbles and release the drug loaded nanofilm on the microbubble membrane in a specific site, so as to realize the liposome nanoparticles in the ultrasonic irradiation field. The effect of medium target to release drugs. Polyethylene glycol (PEG) is a more commonly used modifier in the preparation of lipid system. The introduction of PEG in liposomes can effectively reduce the affinity of the liposome nanoparticles and reduce the phagocytosis of the liposome nanoparticles by the liver macrophages, thus prolonging the circulation time of the liposomes in the blood. At the same time, both liposome nanoparticles and contrast agent microbubbles can provide an effective spatial framework for their respective membranes after the introduction of PEG. On this basis, only a certain modification of PEG can realize the coupling of liposome nanoparticles and contrast agent microbubbles, thus realizing the assumption of the preparation of a new ultrasound contrast agent in this research.
Due to the role of the blood brain barrier, chemotherapeutic drugs are not easy to infiltrate into the meninges, orbital and other "blind areas". Residual leukemic cells in these sites are the main causes of the recurrence of central nervous system leukemia (CNSL). Chemotherapy is the main measure to prevent the recurrence of CNSL, but the later side effects of radiotherapy are obvious and have toxic effects on the endocrine and nervous system. Intrathecal injection of chemotherapeutic drugs is traumatic and easy to cause infection, and large dose of methotrexate (HDMTX) is one of the more effective CNSL prevention methods at present, but there are also large side effects, and also there are many side effects. It can lead to long-term mental toxicity. Therefore, how to achieve effective concentration of MTX in brain tissue and reduce its side effects is the key to the treatment and prevention of CNSL.
The purpose of this study is to prepare the MTX loaded nanoparticles with high efficacy on the basis of the early preparation of "fat fluorine" contrast agent. By using biotin avidin bridging method, the drug loaded nanoparticles and lipid microbubbles were coupled to prepare a new drug carrier contrast agent loaded with MTX nanoparticles, and the safety and release of drugs in the ultrasound irradiation field were observed. At the same time, we discuss the ability of ultrasound combined with coupled MTX nanoparticles to promote the transport of blood brain barrier in rats, and discuss its mechanism preliminarily, so as to explore a new method of non-invasive and safe for the treatment of central nervous system disease.
Methods and routes
1. based on the preparation technology of "lipofluorin", biotinylated "fat fluorine" contrast enhanced ultrasound was prepared.
A certain proportion of biotinylated phosphatidylcholine was added to the original lipid fluorine formula, and the proportion of phospholipid was changed properly. The particle size distribution and concentration of the prepared contrast agent were measured by the Kurt particle size analyzer.
2. preparation of biotinylated MTX lipid nanoparticles based on the preparation technology of "lipofluorin":
The lipid nanoparticles loaded with biotinylated MTX were prepared by two freeze drying and mechanical oscillation method. The separation of drug loaded nanoparticles and free drugs was separated by column chromatography. The encapsulation efficiency of drug loaded nanoparticles was determined by high performance liquid chromatography. The particle size distribution of nanoparticles was measured by Zetasizer3000 and transmission electron microscopy was used for the morphology. The observation and measurement of the particle size distribution.
3. biotinylated "fat fluorine contrast agent" coupled with MTX loaded lipid nanoparticles:
The "liposuction" contrast agent and MTX lipid nanoparticles were prepared with different content of biotinylated biotinylated lipid nanoparticles, and they were coupled by avidin biotin connection system. The unbonded nanoparticles were separated by a rinsing centrifuge method. Finally, the ultrasound contrast agent for the coupling drug nanoparticles was obtained. Coulter Counter should be used to make the coupling drug nanoparticles. The contrast agent was used to determine the size of the particle, and the contrast agent was used to examine the liver of the rat in order to understand the effect of the enhanced imaging, and the MTX carrying capacity of the coupling drug nanoparticles was measured by high performance liquid chromatography.
Inhibition effect and mechanism of 4. lipid loaded MTX lipid nanoparticles on tumor cells in vitro
The tumor cell suspension was irradiated with the coupling of MTX lipid nanoparticles, and the inhibitory rate of the tumor cells was measured by MTT method and apoptosis, and the morphological changes of the cells after the combined action of ultrasound and microbubbles were observed by scanning electron microscopy.
5. ultrasound combined with microbubbles to open blood-brain barrier in rats: an experimental study
Different parameters, including power, irradiation time, duty cycle, and microbubble amount, were selected with the treatment of ultrasound apparatus combined with microbubbles. The Evans-blue (EB) tracer test was used to evaluate the open condition of BBB in rats. The optimum irradiation parameters were screened and the safety and reversibility of the parameters were evaluated.
6. ultrasound combined with drug loaded microbubbles promotes the transport of MTX across the blood-brain barrier in rats:
It was divided into four groups: (1) ultrasound + drug microbubble group; (2) ultrasound + microbubble + drug group; (3) drug carrier microbubble group; (4) drug group. The concentration of MTX in each group was detected by HPLC, and the mechanism was explored by lanthanum nitrate tracer test.
7. the establishment of the model of the blood brain barrier in vitro:
The BALB/c mouse brain astrocytes were cultured in the primary culture, and the Brainmicrovasular endothelial cell (BMVEC) and astrocytes were inoculated in the two sides of the porous membrane of the Transwell chamber, respectively. The transmembrane resistance measurement, permeability test, immunohistochemical ZO-1 detection and transmission electron microscopy evaluation were used. Its morphology and limitation of permeability function.
Results and conclusions
1. the application of freeze-drying technology can successfully prepare biotinylated ultrasound contrast agent, and the preparation of methotrexate lipid nanoparticles can be achieved by using two freeze-drying techniques. The preparation process is relatively simple.
2. Sephadex gel column chromatography can effectively separate drug loaded nanoparticles from free drugs.
The 3. avidin biotin connection system can realize the coupling of lipid microbubbles and drug loaded nanoparticles. This method is worthy of the particle size of the drug carrying contrast agent, which is in accordance with the requirements of intravenous injection, with a higher load capacity and a dose of about (4.91 + 0.51) mg/ml..
4. ultrasound coupled with MTX nanoparticle microbubbles can effectively inhibit the proliferation of tumor cells, and its mechanism is related to the formation of pores on the cell surface.
5. using MetronAP-170 ultrasound therapy instrument, the frequency of the probe is 1MHz, the output power is 2.0W/cm2, the duty ratio is 20%, the irradiation time is 5min, the dose of contrast agent is 0.5ml/kg, and the blood brain barrier of rats can be safely and reversible.
6. using the above parameters, ultrasound combined with microbubbles can significantly increase the transport of MTX across the blood brain barrier in rats, and there is a significant difference compared with the other three groups (P0.01). The mechanism may be related to the cavitation effect of the contrast agent microbubbles under the action of ultrasonic wave, which is related to the close connection and opening of the blood brain barrier.
7. the co culture model of cerebral microvascular endothelial cells and astrocytes has the basic characteristics of BBB in morphology, permeability and barrier properties, which can be used for the study of drug transshipment across the blood brain barrier.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R445.1;R741

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