本文選題：微氣泡 + 磁性納米顆粒　； 參考：《東南大學(xué)》2017年博士論文

【摘要】：近年來,隨著微納載體材料的不斷發(fā)展,磁性納米顆粒和微氣泡已分別發(fā)展成為有效的醫(yī)學(xué)影像造影劑和藥物輸送載體系統(tǒng)。融合兩者的優(yōu)點制備的攜帶磁性納米顆粒的磁性微氣泡載體材料,由于兼具優(yōu)良的磁學(xué)特性和聲學(xué)特性在生物醫(yī)學(xué)應(yīng)用領(lǐng)域得到了較廣泛的關(guān)注和研究。而將磁性微氣泡與藥物、抗體、多肽或多糖結(jié)合起來,形成集早期診斷與精確靶向治療于一體的多模式、多功能造影劑,更是目前生物醫(yī)療造影材料的一個發(fā)展趨勢。本論文將超順磁性APTS/γ-Fe203納米顆粒在聚合物微氣泡表面進(jìn)行了可控組裝并深入探討了組裝機理,在對組裝后的磁性微氣泡進(jìn)行膜殼性質(zhì)、多模態(tài)成像性能等方面深入研究的基礎(chǔ)上,將一種兼具靶向與治療功能的RGD-L-TRAIL蛋白與之結(jié)合,構(gòu)建了RGD-L-TRAIL磁性微氣泡分子探針,此探針既能靶向于腫瘤新生血管并誘導(dǎo)腫瘤細(xì)胞凋亡,在病灶部位富集之后,又可以利用微氣泡的聲學(xué)特性進(jìn)行超聲顯影,利用γ-Fe2O3納米顆粒的磁學(xué)特性進(jìn)行磁共振成像,從而達(dá)到了診療一體化的效果。主要在如下幾個方面進(jìn)行了研究:1.對聚合物微氣泡在穩(wěn)定性及粒徑方面進(jìn)行了優(yōu)化,通過端基羧基化修飾有效提高了微氣泡的分散穩(wěn)定性及耦聯(lián)其它活性物質(zhì)的能力,通過不斷優(yōu)化實驗條件,使微氣泡粒徑更加均勻,在保持超聲成像增強能力的同時,更適于體內(nèi)應(yīng)用,為后續(xù)工作打下了良好的基礎(chǔ)。2.通過"表面裝載"的方式在聚合物微氣泡上組裝了不同濃度梯度的APTS/γ-Fe203超順磁性納米顆粒,對組裝機理及微氣泡膜殼結(jié)構(gòu)的變化進(jìn)行了深入探討,對磁性微氣泡的磁性、穩(wěn)定性、磁共振/超聲成像效果進(jìn)行了考察,結(jié)果表明這種表面裝載磁性納米顆粒的磁性微氣泡具有"高裝載量","高磁性",MRI/US顯影效果的"可調(diào)控性"及潛在的耦聯(lián)其它活性物質(zhì)的特點,顯示出了良好的生物醫(yī)學(xué)應(yīng)用潛力。3.在上述磁性微氣泡表面,偶聯(lián)了靶向腫瘤新生血管和誘導(dǎo)腫瘤細(xì)胞凋亡的RGD-L-TRAIL蛋白,形成了磁性微氣泡分子探針。對此探針進(jìn)行了粒徑、磁滯回線、顯微形貌等結(jié)構(gòu)表征及穩(wěn)定性考察;對磁性微氣泡表面偶聯(lián)的RGD-L-TRAIL蛋白進(jìn)行了定量分析,優(yōu)化出了使磁性微氣泡分子探針兼具靈敏性和穩(wěn)定性的"最合適"蛋白偶聯(lián)量。4.采用頻率為3.5MHz的臨床超聲診斷儀,對優(yōu)化后的磁性微氣泡分子探針進(jìn)行了體外超聲成像實驗,結(jié)果表明其可以有效增強超聲成像效果,維持時間大約在5分鐘左右。同時,采用7T磁共振成像儀,對磁性微氣泡分子探針進(jìn)行了體外磁共振成像研究,結(jié)果表明其可以有效降低磁共振成像時的T2信號。從而證實RGD-L-TRAIL磁性微氣泡分子探針具有超聲/磁共振雙模態(tài)造影的功能效果。5.在體外,分別檢測了未偶聯(lián)RGD-L-TRAIL蛋白的磁性微氣泡,偶聯(lián)蛋白的磁性微氣泡及相同濃度的純RGD-L-TRAIL蛋白誘導(dǎo)結(jié)腸癌細(xì)胞COLO-205凋亡的情況,結(jié)果顯示磁性微氣泡分子探針可以很好地誘導(dǎo)COLO-205細(xì)胞凋亡,而相同劑量的未偶聯(lián)RGD-L-TRAIL的磁性微氣泡不具有誘導(dǎo)凋亡的能力。從而可以證實在與磁性微氣泡偶聯(lián)后,RGD-L-TRAIL蛋白保留了誘導(dǎo)細(xì)胞凋亡的活性,且凋亡具有蛋白劑量依賴性。6.建立了裸鼠結(jié)腸癌腫瘤模型,分別以瘤內(nèi)及尾靜脈兩種注射方式,將RGD-L-TRAIL磁性微氣泡分子探針注入裸鼠體內(nèi),用小動物超聲成像系統(tǒng)進(jìn)行了腫瘤部位的超聲成像實驗,并與未偶聯(lián)RGD-L-TRAIL蛋白的磁性微氣泡進(jìn)行比較,結(jié)果發(fā)現(xiàn)在兩種注射方式下,RGD-L-TRAIL磁性微氣泡分子探針均會使腫瘤部位超聲圖像具有更高的灰度值,且維持更長的灰度保持時間。同時,采用7T磁共振成像儀進(jìn)行了腫瘤部位的磁共振成像實驗,結(jié)果表明,無論用何種注射方式,RGD-L-TRAIL磁性微氣泡分子探針引起的T2信號強度變化均大于無蛋白偶聯(lián)的磁性微氣泡。以上結(jié)果證實了磁性微氣泡分子探針在體內(nèi)可以靶向腫瘤細(xì)胞,更有效地同時增強超聲/磁共振雙模態(tài)成像效果。此外,還通過對腫瘤部位組織切片中Fe及RGD-L-TRAIL蛋白的檢測,進(jìn)一步驗證了磁性納米顆粒、微氣泡和RGD-L-TRAIL蛋白的有效耦聯(lián),及磁性微氣泡分子探針的靶向性。7.最后,考察了 RGD-L-TRAIL磁性微氣泡分子探針體內(nèi)抑制腫瘤的效果。結(jié)果表明,尾靜脈注射RGD-L-TRAIL蛋白及RGD-L-TRAIL磁性微氣泡分子探針均可抑制結(jié)腸癌移植瘤的生長,腫瘤抑制率分別為44.6%和47.5%,而未偶聯(lián)蛋白的磁性微氣泡無法有效抑制腫瘤生長,這一結(jié)果寫TDNEL法檢測各組動物腫瘤細(xì)胞調(diào)亡情況相一致。
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.35

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