本文選題：血腦屏障 + 超聲�。� 參考：《第三軍醫(yī)大學(xué)》2013年博士論文

【摘要】：背景和目的 血腦屏障(Blood brain barrier，BBB)對維持中樞神經(jīng)系統(tǒng)內(nèi)環(huán)境的穩(wěn)定具有重要作用，也正是由于其本身結(jié)構(gòu)功能的特點, BBB阻礙了許多治療中樞神經(jīng)系統(tǒng)疾病藥物的進入，無論是口服還是經(jīng)血管給藥，藥物都很難通過BBB從而進入腦組織，無法在腦組織內(nèi)達到有效治療濃度而實現(xiàn)其治療作用，影響治療效果。研究表明，治療中樞神經(jīng)系統(tǒng)疾病藥物的療效主要是取決于血腦屏障對該藥物的通透性。直接腦室穿刺給藥有創(chuàng)傷性且容易伴發(fā)感染，限制了其應(yīng)用；而對藥物進行相關(guān)或一定修飾、改變分子量大小和電荷，針對葡萄糖、氨基酸、肽類等通過BBB的特殊載體，因給藥效率低，目前還難以廣泛應(yīng)用于臨床。 因此，如何無創(chuàng)、可逆、靶向地開放BBB，使治療藥物透過血腦屏障，在腦區(qū)達到藥物有效治療濃度是目前國內(nèi)外學(xué)者普遍關(guān)注的熱點之一。超聲聯(lián)合微泡輻照腦組織，已證實可以實現(xiàn)可逆、無創(chuàng)性增強血腦屏障通透性，其開放血腦屏障的機制可能與空化效應(yīng)有關(guān)。目前，應(yīng)用超聲波聯(lián)合新型造影劑微泡進行藥物跨BBB轉(zhuǎn)運也為超聲治療開創(chuàng)了新的平臺，超聲微泡造影劑可用作藥物運載體，將藥物包載于造影劑微泡中防止藥物在體循環(huán)中降解失活，結(jié)合超聲靶向微泡擊破（ultra-sound targetedmicrobubble destroy，UTMD）技術(shù)，可以選擇性的將藥物在目的區(qū)域釋放。利用該方法，一方面增加了藥物的局部作用濃度，另一方面也降低了血液中的藥物濃度，從而實現(xiàn)在減少全身性副作用的同時提高藥物療效。 “脂氟顯”是我科自行研制并具有獨立自主知識產(chǎn)權(quán)的新型脂膜超聲造影劑，經(jīng)過前期大量動物實驗研究證實，無論是在診斷還是在治療領(lǐng)域，均取得良好的應(yīng)用研究結(jié)果。但載藥量較低也成為我們將應(yīng)用“脂氟顯”于治療領(lǐng)域的一個障礙，同時回顧目前載藥物或載基因超聲造影劑的相關(guān)研究，超聲造影劑作為藥物或基因載體均有其不可避免的局限性，一方面，造影劑微泡外殼為較薄的脂膜或蛋白膜，在膜上能攜帶的藥物量及基因量均有限，通過透射電鏡對“脂氟顯”進行檢測也證實其外殼由非常薄的膜構(gòu)成；另一方面，造影劑微泡中心為惰性氣體所占據(jù)，這就更減少了造影劑微泡攜帶藥物及基因的能力。因此如何提高超聲造影劑微泡的載藥量成為目前急待解決的問題。 納米微粒是近年來研究較多的新型藥物載體，其載藥的有效性及其控制釋放藥物能力在大量研究中得到了證實，尤其是脂質(zhì)體載藥納米粒，由雙層脂膜中心包裹水性核構(gòu)成，其脂膜上具有攜帶親脂性藥物及兩親性藥物，而中心部的液性部分亦可攜帶親水性藥物，因此其攜載藥物的種類較造影劑微泡更廣泛。同時，脂質(zhì)體作為藥物載體能降低藥物的給藥劑量，減輕藥物毒性，提高藥物的穩(wěn)定性。研究表明，載藥脂質(zhì)體能使藥物在腫瘤部位聚集達到單純應(yīng)用藥物的50~100倍。但要達到不同部位靶向釋放，則需要不同修飾方法進行脂質(zhì)體的制備。若脂質(zhì)體能在超聲波作用下實現(xiàn)目的治療區(qū)的靶向釋放則可減少制備工藝上的煩瑣過程，然而，由于脂質(zhì)體的核心為液態(tài)物質(zhì)，經(jīng)注射入血液后并不能與超聲波產(chǎn)生協(xié)同作用的效果，因此超聲亦無法對納米粒實現(xiàn)實時的監(jiān)測，對于其是否到達目的治療區(qū)亦無從掌握。 基于上述“脂氟顯”及載藥納米粒的相關(guān)特點及其不足，本研究擬在現(xiàn)有制備“脂氟顯”的基礎(chǔ)上，于其表面耦連上載藥脂質(zhì)體納米粒，由于脂質(zhì)體納米粒粒徑較小，造影劑微泡耦連上納米粒后，其粒徑的總體變化不會發(fā)生太大的改變，即超聲造影劑微泡在超聲波聲場中的相關(guān)物理特性不會發(fā)生改變，這樣我們就可以解決造影劑微泡載藥量低的缺陷，同時利用超聲波能有效擊破微泡從而在特定部位釋放微泡膜上耦連的載藥納米粒，實現(xiàn)脂質(zhì)體納米粒在超聲輻照場中靶向釋放藥物的的效果。聚乙二醇(PEG)是脂質(zhì)體制備中較常用的修飾材料，脂質(zhì)體中引入PEG后可有效地降低調(diào)理素對脂質(zhì)體納米粒的親和性，減少肝臟巨噬細胞對載藥脂質(zhì)體納米粒的吞噬作用，從而延長脂質(zhì)體在血液中的循環(huán)時間，同時，無論是脂質(zhì)體納米粒還是造影劑微泡在引入PEG后均能為各自的膜構(gòu)成上提供有效的空間構(gòu)架，在此基礎(chǔ)上僅對PEG進行一定的修飾即可實現(xiàn)脂質(zhì)體納米粒與造影劑微泡的耦連，從而實現(xiàn)本研究中制備新型超聲造影劑的設(shè)想。 由于血腦屏障的作用，化療藥物不易滲入腦膜、眼眶等“盲區(qū)”，這些部位的殘留白血病細胞是造成中樞神經(jīng)系統(tǒng)白血病(central nervous system leukemia, CNSL)復(fù)發(fā)的主要根源。目前，顱腦放療、鞘內(nèi)注射化療藥物和大劑量甲氨蝶呤（Methotrexate,MTX）化療是預(yù)防CNSL復(fù)發(fā)的主要措施，但放療的后期副作用明顯，對內(nèi)分泌、神經(jīng)系統(tǒng)均有毒性作用；鞘內(nèi)注射化療藥物具有創(chuàng)傷性，且易引發(fā)感染；大劑量甲氨蝶呤(HDMTX)是目前較為有效的CNSL預(yù)防方法之一，但亦存在較大的副作用，同時還可以導(dǎo)致遠期的精神毒性。為此，如何使MTX在腦組織病灶內(nèi)達到有效濃度，并降低其毒副作用，是治療和預(yù)防CNSL的關(guān)鍵所在。 本研究目的是在前期制備“脂氟顯”造影劑的基礎(chǔ)上，制備高效載藥的載MTX納米粒，利用生物素-親和素橋接法，將載藥納米粒與脂質(zhì)微泡耦連，制得耦連載MTX納米粒的新型載藥造影劑，，并觀察其安全性及在超聲輻照場中體外釋放藥物的效果；同時探討治療超聲聯(lián)合耦連載MTX納米粒造影劑促藥物跨大鼠血腦屏障轉(zhuǎn)運的能力，并對其機理進行初步探討，為中樞神經(jīng)系統(tǒng)疾病治療探索一種無創(chuàng)、安全的新方法。 方法及路線 1.在“脂氟顯”制備工藝基礎(chǔ)上，制備生物素化“脂氟顯”超聲造影： 將一定比例生物素化磷脂加入原“脂氟顯”脂質(zhì)配方中，適當改變磷脂配比，以庫爾特粒度計數(shù)儀對制備的造影劑進行粒徑分布及濃度進行測定。 2.在“脂氟顯”制備工藝基礎(chǔ)上，制備生物素化載MTX脂質(zhì)納米粒： 利用二次冷凍干燥法及機械振蕩法制備生物素化載MTX的脂質(zhì)納米粒，應(yīng)用柱層析法實現(xiàn)載藥納米粒與游離藥物的分離，應(yīng)用高效液相色譜法對載藥納米粒進行包封率測定，應(yīng)用Zetasizer3000對納米粒進行粒徑分布測定，透射電鏡進行形態(tài)學(xué)及粒徑分布觀察與測量。 3.生物素化“脂氟顯”造影劑與載MTX脂質(zhì)納米粒的耦連： 制備不同含量生物素化的“脂氟顯”造影劑及載MTX脂質(zhì)納米粒，利用親和素－生物素連接體系對兩者進行耦連，以漂洗－離心法分離未結(jié)合的納米粒，最終得到耦連載藥納米粒的超聲造影劑，應(yīng)用Coulter Counter對耦連載藥納米粒超聲造影劑進行粒徑測定；利用制備的造影劑對大鼠肝臟進行造影檢查，以了解其增強顯像效果；應(yīng)用高效液相色譜法測定耦連載藥納米粒造影劑MTX攜帶量。 4.耦連載MTX脂質(zhì)納米粒微泡體外對腫瘤細胞抑制效果及機制研究： 以一定功率治療超聲儀，輻照加入耦連載MTX脂質(zhì)納米粒微泡后的腫瘤細胞懸液，應(yīng)用MTT法及凋亡檢測手段測量其對腫瘤細胞的抑制率，并應(yīng)用掃描電鏡觀察超聲與微泡聯(lián)合作用后細胞形態(tài)變化，以初步了解其作用機制。 5.超聲聯(lián)合微泡開放大鼠血腦屏障的實驗研究： 應(yīng)用治療超聲儀聯(lián)合微泡輻照大鼠顱腦，選擇不同參數(shù)，包括功率、輻照時間、占空比、微泡用量等，以伊文思藍(Evans-blue, EB)示蹤實驗評價大鼠BBB開放情況，篩選出最佳輻照參數(shù)，并評價其安全性及可逆性。 6.超聲聯(lián)合載藥微泡促MTX跨大鼠血腦屏障轉(zhuǎn)運的實驗研究： 分為四組：(1)超聲+載藥微泡組；(2)超聲+微泡+藥物組；(3)載藥微泡組；(4)藥物組。利用高效液相色譜法檢測各組腦組織MTX濃度，并通過硝酸鑭示蹤實驗對其機制進行初探。 7.體外血腦屏障模型的建立： 原代培養(yǎng)BALB/c小鼠腦星形膠質(zhì)細胞，將小鼠腦微血管內(nèi)皮細胞(Brainmicrovasular endothelial cell，BMVEC)與星形膠質(zhì)細胞分別接種于transwell小室多孔濾膜的兩面進行共培養(yǎng)，通過跨膜電阻測量，通透性測試，免疫組化ZO-1檢測，透射電鏡檢查評價其形態(tài)學(xué)及限制通透功能。 結(jié)果及結(jié)論 1.應(yīng)用凍干技術(shù)可以成功制備生物素化超聲造影劑，利用二次凍干技術(shù)可以實現(xiàn)載甲氨蝶呤脂質(zhì)納米粒的制備，該制備方法工藝流程相對簡單。 2.葡聚糖凝膠柱層析法可以有效實現(xiàn)載藥納米粒與游離藥物的分離。 3．親和素－生物素連接體系可以實現(xiàn)脂質(zhì)微泡與載藥納米粒的耦連，此法值得的載藥造影劑粒徑符合靜脈注射要求，有較高載藥量，載藥量約(4.91±0.51) mg/ml。 4.超聲聯(lián)合耦連載MTX納米粒微泡能有效抑制腫瘤細胞的增殖，其作用機制與其在細胞表面形成孔洞有關(guān)。 5.采用MetronAP-170超聲波治療儀，探頭頻率1MHz，輸出功率2.0W/cm2、占空比20%、輻照時間5min、造影劑劑量0.5ml/kg，可以安全可逆的開放大鼠血腦屏障。 6.采用上述參數(shù)，超聲聯(lián)合載藥微泡可以明顯增加MTX跨大鼠血腦屏障轉(zhuǎn)運，與其余三組比較有顯著性差異(P0.01)，其機制可能與造影劑微泡在超聲波的作用下產(chǎn)生的空化作用，引起了血腦屏障緊密連接開放有關(guān)。 7．腦微血管內(nèi)皮細胞與星形膠質(zhì)細胞共培養(yǎng)模型，在形態(tài)學(xué)，通透性及屏障性方面具備了BBB的基本特征，可用于藥物跨血腦屏障轉(zhuǎn)運的研究。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R445.1;R741

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