基于CNTs載藥性和靶向性的新型超聲造影劑的研究
發(fā)布時間：2018-11-15 17:22
【摘要】：目的基于碳納米管載藥性和靶向性設(shè)計制備一種集靶向、化療于一體的新型多功能超聲造影劑。通過研究制備復(fù)合微泡的工藝以及微泡中碳納米管和紫杉醇的負(fù)載率,探索其體外抗腫瘤活性、體內(nèi)外超聲顯像效果以及體內(nèi)急性毒性;系統(tǒng)地對復(fù)合微泡進(jìn)行臨床前研究,為這種新型多功能超聲造影劑的臨床應(yīng)用提供了基礎(chǔ)研究數(shù)據(jù)。方法(1)采用聲振空化法,以司盤-聚乙二醇(Span-PEG)為膜材,加入葉酸-碳納米管-紫杉醇(folate-carbon nanotube-paclitaxel,FA-CNTs-PTX)復(fù)合物,制備復(fù)合FA-CNTs-PTX的司盤-聚乙二醇(Span-PEG)微泡超聲造影劑;通過紅外光譜儀、紫外分光光度計、熱重儀、掃描電鏡、透射電鏡及激光粒度分析儀等對微泡結(jié)構(gòu)和形貌進(jìn)行表征分析。(2)采用多普勒超聲診斷儀,分別考察復(fù)合微泡體外和體內(nèi)超聲造影效果。(3)選取乳腺癌MCF-7細(xì)胞、肝癌HepG2細(xì)胞和正常人臍靜脈內(nèi)皮HUVEC細(xì)胞作為試驗(yàn)?zāi)Ｐ?采用CCK-8試劑盒及AO/EB試劑盒雙染色法檢測復(fù)合微泡超聲造影劑體外抗腫瘤活性。通過流式細(xì)胞術(shù)定量分析復(fù)合微泡對乳腺癌MCF-7細(xì)胞和正常人臍靜脈內(nèi)皮HUVEC細(xì)胞的凋亡率。(4)以昆明小鼠為模型,通過急性毒性試驗(yàn)確定復(fù)合微泡超聲造影劑對小鼠的最大耐受量。結(jié)果(1)所制備的復(fù)合FA-CNTs-PTX的Span-PEG微泡表面光滑、平均粒徑為442 nm,微泡中碳納米管和紫杉醇的負(fù)載率分別為1.69%和47.9%。成功將FA-CNTsPTX靶向載藥復(fù)合物包覆于Span-PEG微泡中,微泡為空心球體,粒徑分布均勻且為納米級別。(2)與生理鹽水和Span-PEG微泡相比,復(fù)合FA-CNTs-PTX微泡超聲造影劑在體外和體內(nèi)均具有更顯著的增強(qiáng)超聲信號的性質(zhì),并且超聲成像的清晰度有所提高。(3)復(fù)合FA-CNTs-PTX的微泡超聲造影劑對乳腺癌MCF-7細(xì)胞有明顯的增殖抑制作用,且主要取決于較高的載藥率及微泡的納米尺寸;復(fù)合FA-CNTs-PTX的微泡超聲造影劑能夠影響乳腺癌MCF-7細(xì)胞的增殖抑制率及細(xì)胞的凋亡周期,并對細(xì)胞MCF-7的增殖抑制作用程度高于肝癌HepG2細(xì)胞;復(fù)合FA-CNTs-PTX的微泡超聲造影劑對乳腺癌MCF-7細(xì)胞的早期凋亡率34.85%遠(yuǎn)大于對正常人臍靜脈內(nèi)皮HUVEC細(xì)胞的早期凋亡率14.99%,復(fù)合FA-CNTs-PTX的微泡超聲造影劑對乳腺癌MCF-7細(xì)胞具有明顯的靶向抑制作用。(4)最大耐受劑量下昆明小鼠沒有出現(xiàn)死亡及不良反應(yīng),最大耐受劑量為50 kg成人的350倍。結(jié)論本研究設(shè)計和制備的復(fù)合FA-CNTs-PTX微泡超聲造影劑有望成為一種良好的集造影和靶向治療為一體的超聲造影劑。首先,復(fù)合微泡的結(jié)構(gòu)滿足作為造影劑的理論條件,在體內(nèi)外均具有理想的增強(qiáng)超聲顯影功能;其次,該復(fù)合微泡成功負(fù)載紫杉醇藥物,實(shí)現(xiàn)靶向抑制乳腺癌MCF-7細(xì)胞增殖作用。最后,復(fù)合微泡作用于昆明小鼠的最大耐受劑量已經(jīng)遠(yuǎn)高于成人用量的百倍,所以復(fù)合FA-CNTs-PTX微泡超聲造影劑在體內(nèi)安全穩(wěn)定,且提供比較理想的超聲顯影效果,是一種高效的新輔助化療試劑,為下一步體內(nèi)遞藥提供研究的藥理模型。
[Abstract]:Objective To prepare a novel multi-functional ultrasound contrast agent integrated with target and chemotherapy based on the properties of carbon nanotubes and the targeted design. The method for preparing the composite micro-bubble and the loading rate of the carbon nano-tube and the paclitaxel in the micro-bubble are researched, the in vitro anti-tumor activity, the in-vivo ultrasound imaging effect and the in-vivo acute toxicity are explored, The basic research data is provided for the clinical application of the novel multifunctional ultrasound contrast agent. The method comprises the following steps: (1) adopting an acoustic vibration cavitation method, taking the pan-polyethylene glycol (Span-PEG) as a membrane material, adding a folic acid-carbon nano-tube-paclitaxel (FA-CNTs-PTX) complex, and preparing a Si-polyethylene glycol (Span-PEG) micro-bubble ultrasonic contrast agent of the composite FA-CNTs-PTX; and through an infrared spectrometer, The micro-bubble structure and morphology were characterized and analyzed by ultraviolet spectrophotometer, thermogravimeter, scanning electron microscope, transmission electron microscope and laser particle size analyzer. (2) Doppler ultrasonic diagnostic apparatus was used to study the effect of combined microvesicle in vitro and in vivo. (3) The anti-tumor activity of human breast cancer MCF-7 cells, liver cancer HepG2 cells and human umbilical vein endothelial HUVEC cells was selected as the test model, and the anti-tumor activity of the composite micro-bubble ultrasound contrast agent was detected by using the CCK-8 kit and the AO/ EB kit double staining method. The apoptosis rate of HUVEC cells in human umbilical vein endothelial cells (MCF-7) and human umbilical vein endothelial cells (HUVEC) was analyzed by flow cytometry. (4) The maximum tolerance of the compound micro-bubble ultrasound contrast agent to the mice was determined by acute toxicity test with Kunming mice as the model. Results (1) The surface of the Span-PEG microfoam of the composite FA-CNTs-PTX was smooth, the average particle size was 442 nm, and the loading rates of the carbon nanotubes and the paclitaxel in the micro-bubbles were 1.69% and 44.9%, respectively. The FA-CNTPTX-targeting drug-containing compound was successfully coated in the Span-PEG micro-bubble, and the micro-bubble is a hollow sphere, and the particle size distribution is uniform and the nanometer level is nanometer. (2) Compared with the normal saline and the Span-PEG microvesicle, the composite FA-CNTs-PTX micro-bubble ultrasound contrast agent has more significant enhancement of the nature of the ultrasound signal both in vitro and in vivo, and the clarity of the ultrasound imaging is improved. and (3) the micro-bubble ultrasound contrast agent of the compound FA-CNTs-PTX has obvious proliferation inhibition effect on the breast cancer MCF-7 cells, and mainly depends on the higher drug loading rate and the nano size of the micro-bubbles; and the micro-bubble ultrasound contrast agent of the composite FA-CNTs-PTX can influence the proliferation inhibition rate of the breast cancer MCF-7 cells and the apoptosis cycle of the cells, The inhibition of the proliferation of the cell line MCF-7 was higher than that of the HepG2 cells. The early apoptotic rate of the composite FA-CNTs-PTX was 34. 85%, which was much higher than that of the human umbilical vein endothelial HUVEC cells. The micro-bubble ultrasound contrast agent of compound FA-CNTs-PTX has obvious targeted inhibition on breast cancer MCF-7 cells. (4) No death and adverse reaction occurred in Kunming mice at the maximum tolerated dose, and the maximum tolerated dose was 350 times of that of 50 kg of adult. Conclusion The complex FA-CNTs-PTX micro-bubble ultrasound contrast agent designed and prepared in this study is expected to be a good contrast agent for both contrast and targeted therapy. firstly, the structure of the composite micro-bubble meets the theoretical condition as a contrast agent, and has an ideal enhanced ultrasonic developing function in the outside of the body; secondly, the composite micro-bubble is successfully loaded with the paclitaxel medicament to realize the targeted inhibition of the proliferation of the breast cancer MCF-7 cell. and finally, the maximum tolerated dose of the composite micro-bubble on the Kunming mouse is much higher than that of the adult, so the composite FA-CNTs-PTX micro-bubble ultrasonic contrast agent is safe and stable in the body, The pharmacological model of the study was provided for the next in-vivo delivery.
【學(xué)位授予單位】：佳木斯大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R943
[Abstract]:Objective To prepare a novel multi-functional ultrasound contrast agent integrated with target and chemotherapy based on the properties of carbon nanotubes and the targeted design. The method for preparing the composite micro-bubble and the loading rate of the carbon nano-tube and the paclitaxel in the micro-bubble are researched, the in vitro anti-tumor activity, the in-vivo ultrasound imaging effect and the in-vivo acute toxicity are explored, The basic research data is provided for the clinical application of the novel multifunctional ultrasound contrast agent. The method comprises the following steps: (1) adopting an acoustic vibration cavitation method, taking the pan-polyethylene glycol (Span-PEG) as a membrane material, adding a folic acid-carbon nano-tube-paclitaxel (FA-CNTs-PTX) complex, and preparing a Si-polyethylene glycol (Span-PEG) micro-bubble ultrasonic contrast agent of the composite FA-CNTs-PTX; and through an infrared spectrometer, The micro-bubble structure and morphology were characterized and analyzed by ultraviolet spectrophotometer, thermogravimeter, scanning electron microscope, transmission electron microscope and laser particle size analyzer. (2) Doppler ultrasonic diagnostic apparatus was used to study the effect of combined microvesicle in vitro and in vivo. (3) The anti-tumor activity of human breast cancer MCF-7 cells, liver cancer HepG2 cells and human umbilical vein endothelial HUVEC cells was selected as the test model, and the anti-tumor activity of the composite micro-bubble ultrasound contrast agent was detected by using the CCK-8 kit and the AO/ EB kit double staining method. The apoptosis rate of HUVEC cells in human umbilical vein endothelial cells (MCF-7) and human umbilical vein endothelial cells (HUVEC) was analyzed by flow cytometry. (4) The maximum tolerance of the compound micro-bubble ultrasound contrast agent to the mice was determined by acute toxicity test with Kunming mice as the model. Results (1) The surface of the Span-PEG microfoam of the composite FA-CNTs-PTX was smooth, the average particle size was 442 nm, and the loading rates of the carbon nanotubes and the paclitaxel in the micro-bubbles were 1.69% and 44.9%, respectively. The FA-CNTPTX-targeting drug-containing compound was successfully coated in the Span-PEG micro-bubble, and the micro-bubble is a hollow sphere, and the particle size distribution is uniform and the nanometer level is nanometer. (2) Compared with the normal saline and the Span-PEG microvesicle, the composite FA-CNTs-PTX micro-bubble ultrasound contrast agent has more significant enhancement of the nature of the ultrasound signal both in vitro and in vivo, and the clarity of the ultrasound imaging is improved. and (3) the micro-bubble ultrasound contrast agent of the compound FA-CNTs-PTX has obvious proliferation inhibition effect on the breast cancer MCF-7 cells, and mainly depends on the higher drug loading rate and the nano size of the micro-bubbles; and the micro-bubble ultrasound contrast agent of the composite FA-CNTs-PTX can influence the proliferation inhibition rate of the breast cancer MCF-7 cells and the apoptosis cycle of the cells, The inhibition of the proliferation of the cell line MCF-7 was higher than that of the HepG2 cells. The early apoptotic rate of the composite FA-CNTs-PTX was 34. 85%, which was much higher than that of the human umbilical vein endothelial HUVEC cells. The micro-bubble ultrasound contrast agent of compound FA-CNTs-PTX has obvious targeted inhibition on breast cancer MCF-7 cells. (4) No death and adverse reaction occurred in Kunming mice at the maximum tolerated dose, and the maximum tolerated dose was 350 times of that of 50 kg of adult. Conclusion The complex FA-CNTs-PTX micro-bubble ultrasound contrast agent designed and prepared in this study is expected to be a good contrast agent for both contrast and targeted therapy. firstly, the structure of the composite micro-bubble meets the theoretical condition as a contrast agent, and has an ideal enhanced ultrasonic developing function in the outside of the body; secondly, the composite micro-bubble is successfully loaded with the paclitaxel medicament to realize the targeted inhibition of the proliferation of the breast cancer MCF-7 cell. and finally, the maximum tolerated dose of the composite micro-bubble on the Kunming mouse is much higher than that of the adult, so the composite FA-CNTs-PTX micro-bubble ultrasonic contrast agent is safe and stable in the body, The pharmacological model of the study was provided for the next in-vivo delivery.
【學(xué)位授予單位】：佳木斯大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R943
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