多西紫杉醇靶向納米藥物對(duì)小細(xì)胞肺癌治療作用的研究
發(fā)布時(shí)間：2018-08-23 14:41
【摘要】：流行病學(xué)統(tǒng)計(jì)結(jié)果顯示,每年因癌癥而死亡的病例中約有26%的人死于肺癌,而約15%的新增肺癌病例是小細(xì)胞肺癌(Small Cell Lung Cancer, SCLC)。小細(xì)胞肺癌是肺癌中惡性程度最高的腫瘤之一,臨床治療多以化療為主,初治有效率高,但治療過(guò)程中極易發(fā)生繼發(fā)性耐藥,而且治療后易復(fù)發(fā),患者的兩年生存率極低。因此,人們迫切需要找到針對(duì)小細(xì)胞肺癌治療的有效化療藥物。隨著納米技術(shù)的發(fā)展,越來(lái)越多的納米藥物被應(yīng)用到腫瘤的靶向治療中,如脂質(zhì)體、樹(shù)枝狀分子、聚合物膠束、超順磁氧化鐵晶體和膠體金藥物。已知納米藥物遞送涉及到被動(dòng)靶向和主動(dòng)靶向。被動(dòng)靶向是基于腫瘤周圍脈管系統(tǒng)增強(qiáng)的滲透和滯留效應(yīng)進(jìn)行藥物遞送,無(wú)法從根本上克服常規(guī)化療藥物的非選擇性殺傷作用。主動(dòng)靶向則主要依賴配體與腫瘤細(xì)胞表面的受體結(jié)合,從而“引導(dǎo)”納米粒子進(jìn)入腫瘤細(xì)胞�，F(xiàn)在已經(jīng)開(kāi)發(fā)的配體主要有葉酸、肽、蛋白、抗體、適配子和寡核苷酸。目前已有靶向納米藥物輸送系統(tǒng)應(yīng)用于多種腫瘤的治療報(bào)道,有些藥物甚至已經(jīng)進(jìn)入臨床試驗(yàn),但小細(xì)胞肺癌靶向遞藥系統(tǒng)的研究卻鮮有報(bào)道。本研究旨在構(gòu)建多西紫杉醇靶向納米藥物并評(píng)價(jià)其治療效應(yīng)。我們以SCLC細(xì)胞系H446為靶分子,采用噬菌體展示技術(shù)獲得了與SCLC細(xì)胞系特異性結(jié)合的七肽。在采用乳化溶劑揮發(fā)法構(gòu)建包載多西紫杉醇納米粒子的基礎(chǔ)上,將其與靶向肽相連接制備靶向納米制劑。最后,通過(guò)體內(nèi)外實(shí)驗(yàn)探究其抗腫瘤作用及其作用機(jī)制。主要研究結(jié)果如下：1. 以SCLC細(xì)胞系H446為靶分子,利用噬菌體展示技術(shù)篩選出能與之特異性結(jié)合的七肽(AP和AF)。并采用免疫組織化學(xué)技術(shù)評(píng)價(jià)其特異性。結(jié)果顯示,篩選出的七肽具有與人小細(xì)胞肺癌病理組織特異性結(jié)合的能力。2.采用可生物降解的高分子材料聚乳酸(PLA)包載多西紫杉醇制備納米藥物,然后利用EDC縮合反應(yīng)將其與靶向肽連接構(gòu)建靶向納米藥物。理化性質(zhì)分析結(jié)果顯示,制備的納米制劑平均粒徑為195.4±3.9 nm,PDI平均值為0.19±0.03,平均Zeta電位值為-30.5±0.9 mV,而靶向納米制劑的平均粒徑為285.9±4.4 nm,PDI平均值為0.148±0.036,平均Zeta電位值為-30.1±0.6 mV. TEM電鏡結(jié)果顯示,靶向納米粒子表面光滑,粒徑均一。體外釋放結(jié)果表明納米藥物能夠有效釋放。3.成功制備靶向納米藥物后進(jìn)行體外抗腫瘤作用研究。細(xì)胞毒性實(shí)驗(yàn)結(jié)果顯示,相同濃度的靶向納米藥物對(duì)小細(xì)胞肺癌細(xì)胞系H446的抑制作用顯著強(qiáng)于常規(guī)藥物多西紫杉醇,并具有一定的濃度依賴性。4. 細(xì)胞攝取結(jié)果表明,小細(xì)胞肺癌細(xì)胞系H446對(duì)靶向納米制劑有較好的攝取能力,從而使靶向納米制劑具有更強(qiáng)的殺傷作用。5.在體實(shí)驗(yàn)結(jié)果表明,與多西紫杉醇相比,多西紫杉醇靶向納米制劑能夠顯著地抑制腫瘤生長(zhǎng),且動(dòng)物對(duì)藥物的耐受性良好。6.為探討靶向納米制劑的作用機(jī)制,我們對(duì)其體內(nèi)分布情況進(jìn)行了分析。實(shí)驗(yàn)結(jié)果顯示,靶向納米藥物在腫瘤組織分布量是常規(guī)藥物的4～7倍,而多西紫杉醇在動(dòng)物肝臟及心臟的濃度均顯著高于靶向納米制劑。7. 各組動(dòng)物的心、肝、脾、肺、腎、腦組織HE染色結(jié)果顯示,各劑型藥物均能抑制肺癌肝轉(zhuǎn)移,但多西紫杉醇組和納米藥物組動(dòng)物肝臟均有不同程度的肝細(xì)胞顆粒樣、脂肪樣變形,而靶向納米藥物組動(dòng)物肝臟狀態(tài)良好。綜上所述,我們基于篩選得到的靶向肽構(gòu)建了靶向納米制劑,其在體內(nèi)和體外實(shí)驗(yàn)均表現(xiàn)出明顯優(yōu)于常規(guī)藥物的腫瘤細(xì)胞殺傷作用。靶向納米藥物在腫瘤組織具有較高的藥物分布,此外還能有效地抑制小細(xì)胞肺癌的肝轉(zhuǎn)移。
[Abstract]:Epidemiological statistics show that about 26% of cancer deaths each year are caused by lung cancer, and about 15% of new lung cancer cases are small cell lung cancer (SCLC). With the development of nanotechnology, more and more nano-drugs have been applied to targeted therapy of cancer, such as liposome, dendrimer, polymerization. Biomicelles, superparamagnetic iron oxide crystals and colloidal gold drugs. It is known that nanodrug delivery involves passive targeting and active targeting. Passive targeting is based on enhanced penetration and retention in the peripheral vascular system of the tumor, which can not fundamentally overcome the non-selective killing effect of conventional chemotherapeutic drugs. Active targeting is predominant. Ligands that have been developed are folic acid, peptide, protein, antibody, aptamer and oligonucleotide. Targeted nanodrug delivery systems have been reported to be used in the treatment of various tumors, and some drugs have even entered clinical practice. The aim of this study was to construct docetaxel-targeted nano-drug and evaluate its therapeutic effect. We used the SCLC cell line H446 as the target molecule and obtained the heptapeptides specifically binding to SCLC cell line by phage display technique. The heptapeptides were constructed by emulsion solvent evaporation method. Finally, the antitumor effect and mechanism of docetaxel nanoparticles were investigated by in vitro and in vivo experiments. The main results were as follows: 1. Using SCLC cell line H446 as the target molecule, seven specific binding compounds were screened by phage display technique. Peptides (AP and AF) were evaluated by immunohistochemistry. The results showed that the heptapeptides had the ability to bind specifically to the pathological tissues of human small cell lung cancer (SCLC). 2. Nano-drugs were prepared by biodegradable polymeric material polylactic acid (PLA) encapsulated with docetaxel and then targeted by EDC condensation reaction. The results of physicochemical properties analysis showed that the average particle size, PDI, Zeta potential and PDI were 195.4 (+ 3.9) nm, 0.19 (+ 0.03) and - 30.5 (+ 0.9) mV, respectively, while the average particle size, PDI and Zeta potential were 285.9 (+ 4.4) nm, 0.148 (+ 0.036) and - 30.1 (+ 0.6) mV.TEM. The results of electron microscopy showed that the surface of targeted nanoparticles was smooth and the particle size was uniform. The results of in vitro release showed that the nanoparticles could be effectively released. 3. The antitumor effect of targeted nanoparticles was studied in vitro after the successful preparation of targeted nanoparticles. The results of cell uptake showed that small cell lung cancer cell line H446 had a better uptake of targeted nanoparticles, which made targeted nanoparticles have a stronger killing effect. 5. In vivo results showed that compared with docetaxel, docetaxel had a stronger killing effect. Targeted nanoparticles can significantly inhibit tumor growth and are well tolerated by animals. 6. To investigate the mechanism of targeting nanoparticles, we analyzed the distribution in vivo. The results showed that the distribution of targeted nanoparticles in tumor tissue was 4-7 times as much as that of conventional drugs, while docetaxel in animal liver. HE staining of heart, liver, spleen, lung, kidney and brain tissues showed that all the dosage forms of drugs could inhibit liver metastasis of lung cancer, but the liver of both docetaxel group and nano-drug group had different degrees of hepatocyte granule-like and fatty deformation. In conclusion, we have constructed targeted nanoparticles based on the selected targeting peptides, which exhibit better cytotoxicity than conventional drugs in vivo and in vitro. Targeted nanoparticles have higher drug distribution in tumor tissues and can effectively inhibit small cell lung cancer. Liver metastasis.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2
本文編號(hào)：2199376
[Abstract]:Epidemiological statistics show that about 26% of cancer deaths each year are caused by lung cancer, and about 15% of new lung cancer cases are small cell lung cancer (SCLC). With the development of nanotechnology, more and more nano-drugs have been applied to targeted therapy of cancer, such as liposome, dendrimer, polymerization. Biomicelles, superparamagnetic iron oxide crystals and colloidal gold drugs. It is known that nanodrug delivery involves passive targeting and active targeting. Passive targeting is based on enhanced penetration and retention in the peripheral vascular system of the tumor, which can not fundamentally overcome the non-selective killing effect of conventional chemotherapeutic drugs. Active targeting is predominant. Ligands that have been developed are folic acid, peptide, protein, antibody, aptamer and oligonucleotide. Targeted nanodrug delivery systems have been reported to be used in the treatment of various tumors, and some drugs have even entered clinical practice. The aim of this study was to construct docetaxel-targeted nano-drug and evaluate its therapeutic effect. We used the SCLC cell line H446 as the target molecule and obtained the heptapeptides specifically binding to SCLC cell line by phage display technique. The heptapeptides were constructed by emulsion solvent evaporation method. Finally, the antitumor effect and mechanism of docetaxel nanoparticles were investigated by in vitro and in vivo experiments. The main results were as follows: 1. Using SCLC cell line H446 as the target molecule, seven specific binding compounds were screened by phage display technique. Peptides (AP and AF) were evaluated by immunohistochemistry. The results showed that the heptapeptides had the ability to bind specifically to the pathological tissues of human small cell lung cancer (SCLC). 2. Nano-drugs were prepared by biodegradable polymeric material polylactic acid (PLA) encapsulated with docetaxel and then targeted by EDC condensation reaction. The results of physicochemical properties analysis showed that the average particle size, PDI, Zeta potential and PDI were 195.4 (+ 3.9) nm, 0.19 (+ 0.03) and - 30.5 (+ 0.9) mV, respectively, while the average particle size, PDI and Zeta potential were 285.9 (+ 4.4) nm, 0.148 (+ 0.036) and - 30.1 (+ 0.6) mV.TEM. The results of electron microscopy showed that the surface of targeted nanoparticles was smooth and the particle size was uniform. The results of in vitro release showed that the nanoparticles could be effectively released. 3. The antitumor effect of targeted nanoparticles was studied in vitro after the successful preparation of targeted nanoparticles. The results of cell uptake showed that small cell lung cancer cell line H446 had a better uptake of targeted nanoparticles, which made targeted nanoparticles have a stronger killing effect. 5. In vivo results showed that compared with docetaxel, docetaxel had a stronger killing effect. Targeted nanoparticles can significantly inhibit tumor growth and are well tolerated by animals. 6. To investigate the mechanism of targeting nanoparticles, we analyzed the distribution in vivo. The results showed that the distribution of targeted nanoparticles in tumor tissue was 4-7 times as much as that of conventional drugs, while docetaxel in animal liver. HE staining of heart, liver, spleen, lung, kidney and brain tissues showed that all the dosage forms of drugs could inhibit liver metastasis of lung cancer, but the liver of both docetaxel group and nano-drug group had different degrees of hepatocyte granule-like and fatty deformation. In conclusion, we have constructed targeted nanoparticles based on the selected targeting peptides, which exhibit better cytotoxicity than conventional drugs in vivo and in vitro. Targeted nanoparticles have higher drug distribution in tumor tissues and can effectively inhibit small cell lung cancer. Liver metastasis.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R734.2
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，本文編號(hào)：2199376
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