遞送siRNA的EGFR靶向免疫脂質(zhì)體的制備及其體內(nèi)外活性研究
發(fā)布時間：2018-07-14 21:44
【摘要】：基因治療是目前治療遺傳性疾病或后天獲得性疾病比較理想的治療手段,尤其是惡性腫瘤的治療。RNAi(RNA interfering, RNA干擾)作為一種高效的序列特異性基因沉默技術(shù)在惡性腫瘤基因治療領(lǐng)域發(fā)展掀起了一股研究熱潮,其中,siRNA(small interfering RNA,小干擾RNA)是RNAi路徑中的效應(yīng)分子,它是一種21~23 bp的短片段雙鏈RNA,能夠特異性降解同源序列的mRNA,抑制特異腫瘤相關(guān)基因的表達,從而達到抑制腫瘤生長、侵襲和轉(zhuǎn)移的目的。然而,如果沒有載體的幫助,siRNA無法進入腫瘤細胞內(nèi),載體是制約siRNA基因治療的首要問題,所以,siRNA的遞送載體研究是目前腫瘤基因治療研究的熱點問題。陽離子脂質(zhì)體是目前應(yīng)用較為廣泛的遞送siRNA的一種非病毒載體,它具有無毒、可自然降解、無免疫原性、可以大量合成并放大生產(chǎn)等優(yōu)點,近年來備受研究者的重視。 本課題組前期一直致力于遞送siRNA的陽離子脂質(zhì)體的制備和活性研究,前期研究發(fā)現(xiàn),通過對陽離子脂質(zhì)體進行PEG(聚乙二醇)化、HER2抗體修飾以及冷凍干燥等處理后制備得到一種PEG化免疫凍干脂質(zhì)體(Lyophilized PEGylated Immunoliposomes, LPIL),這種脂質(zhì)體在PEG含量為2.5 mol%時,能有效的將siRNA特異性遞送至高表達HER2乳腺癌細胞內(nèi)并沉默相關(guān)基因的表達。PEG能顯著提高脂質(zhì)體在血漿中的穩(wěn)定性,延長藥物體內(nèi)半衰期,當PEG含量大于8 mol%時,粒徑為100 nm左右的脂質(zhì)體表面呈刷狀,這種刷狀構(gòu)象能完全覆蓋脂質(zhì)體表面,為脂質(zhì)體免于網(wǎng)狀內(nèi)皮系統(tǒng)的吞噬提供更全面的保護。然而LPIL的PEG含量較低,體內(nèi)應(yīng)用受到限制。并且,LPIL無法引入較高含量的PEG,因為較高含量的PEG會破壞LPIL的物理穩(wěn)定性,而且會大大降低siRNA的包封率。所以我們亟待發(fā)展一種高PEG含量、高siRNA包封率以及靶向性較好的陽離子脂質(zhì)體。 LPD(liposome-polycation-DNA,脂質(zhì)體-多聚陽離子-DNA復(fù)合物)是一種新型遞送siRNA的陽離子脂質(zhì)體,它的結(jié)構(gòu)是PEG(聚乙二醇)化的包裹魚精蛋白、siRNA/DNA復(fù)合物的陽離子脂質(zhì)體,與傳統(tǒng)陽離子脂質(zhì)體不同的是,DNA在魚精蛋白的作用下將siRNA壓縮,三者形成一個緊密的帶負電的核,與陽離子脂質(zhì)體混合后通過自組裝過程形成穩(wěn)定的LPD,尤為重要的是,LPD采用后插入PEG的方法在其表面修飾了高含量的PEG,不但保證了siRNA的高包封率,而且有效地增加了脂質(zhì)體的穩(wěn)定性,在體內(nèi)被證明能有效地遞送siRNA至腫瘤細胞中。然而,抗體修飾的LPD尚無人系統(tǒng)探討過其各種納米表征和體內(nèi)外活性。本研究正是在前期PEG化免疫陽離子脂質(zhì)體和LPD的基礎(chǔ)之上,通過一系列的處方篩選,首次優(yōu)化出一種高PEG含量的EGFR靶向免疫脂質(zhì)體TLPD-FCC,并對其各種納米表征及其體內(nèi)外活性進行了較為深入的研究和探討。 首先,我們將DOTAP/Chol陽離子脂質(zhì)體與魚精蛋白、小牛胸腺DNA、siRNA混合得到Naked LPD(非PEG化脂質(zhì)體),然后通過PEG化和引入抗體(Anti-EGFR mAb或Fab’),制備得到EGFR靶向免疫脂質(zhì)體TLPD,針對抗體對脂質(zhì)體粒徑大小以及zeta電位的影響,對抗體類型、連接方式和投入量進行優(yōu)化,結(jié)果發(fā)現(xiàn)抗體類型為Anti-EGFR Fab’,且采用傳統(tǒng)連接方式連接的抗體時制備得到的脂質(zhì)體TLPD-FC,平均粒徑在150 nm~160 nm之間,zeta電位在10 mV左右,為后續(xù)實驗研究奠定了基礎(chǔ)。 然后,通過SDS-PAGE實驗證實抗體確實已經(jīng)連接到脂質(zhì)體,同時考察了Naked-LPD、NTLPD(PEG化非靶向脂質(zhì)體)、TLPD-FC對siRNA結(jié)合能力、siRNA的包封率以及體外基因沉默效率。凝膠阻滯實驗結(jié)果表明Naked-LPD、NTLPD、TLPD-FC對siRNA均具有較強的結(jié)合能力,通過超濾離心的方法證實了siRNA包封率高達90%,以上實驗證實上述樣品對siRNA很強的包裹能力,并且PEG化或者抗體修飾對siRNA包封率影響較小。體外基因沉默效率考查了抗體投入量不同時TLPD-FC(包括TLPD-FCA、TLPD-FCB、TLPD-FCC、TLPD-FCD)在MDA-MB-231細胞中的基因沉默效率,結(jié)果表明TLPD-FCC具有最高的基因沉默效率。 最后,對TLPD-FCC的相關(guān)性質(zhì)和體內(nèi)外活性進行了深入的研究和探討。通過透射電鏡觀察TLPD-FCC和NTLPD的形態(tài)大小發(fā)現(xiàn),兩者在形態(tài)和大小分布上沒有明顯區(qū)別,表明了抗體連接對脂質(zhì)體的結(jié)構(gòu)幾乎沒有影響。瓊脂糖凝膠電泳實驗證實了siRNA在TLPD-FCC或NTLPD的保護下血清穩(wěn)定性良好。脂質(zhì)體的血清穩(wěn)定性通過動態(tài)光散射實驗得到結(jié)論:與Naked LPD相比,NTLPD或TLPD-FCC在PEG的保護下不易與BSA相互作用,穩(wěn)定性好。體外基因轉(zhuǎn)染效率和基因沉默效率結(jié)果表明與NTLPD相比,TLPD-FCC具有較高的特異性的轉(zhuǎn)染效率和基因沉默活性。隨后,通過MDA-MB-231乳腺癌腫瘤模型的建立,免疫熒光標記實驗證實了體內(nèi)腫瘤細胞的EGFR表達水平,體內(nèi)分布實驗也驗證了TLPD-FCC通過受體介導(dǎo)內(nèi)吞機制,隨著時間的變化在腫瘤部位高度聚集,達到峰值,且激光共聚焦結(jié)果顯示,TLPD-FCC顯示出比NTLPD更高的腫瘤細胞靶向特異性和結(jié)合能力及內(nèi)吞效率。最終,體內(nèi)基因沉默效率的考察結(jié)果表明TLPD-FCC具有比NTLPD更高的基因沉默效率,具有特異性的基因沉默活性。 本研究制備得到的TLPD-FCC能有效地遞送siRNA至高表達EGFR的乳腺癌細胞,并具有良好的體內(nèi)外基因沉默效率,有可能作為一種治療高表達EGFR乳腺癌的基因載體用于臨床。
[Abstract]:Gene therapy is an ideal treatment for the treatment of hereditary diseases or acquired acquired diseases, especially the treatment of.RNAi (RNA interfering, RNA interference), as an efficient sequence specific gene silencing technique in the field of malignant tumor gene therapy, set off a hot research upsurge, of which, siRNA (small in). Terfering RNA, small interference RNA) is an effector in the RNAi path. It is a short segment double chain RNA of 21~23 BP, which can specifically degrade mRNA of the homologous sequence, inhibit the expression of specific tumor related genes, and thus inhibit the growth, invasion and metastasis of tumor. But, siRNA can not enter the tumor fine without the help of the carrier. In the cell, the carrier is the most important problem that restricts the siRNA gene therapy. Therefore, the research on the delivery carrier of siRNA is a hot issue in the research of tumor gene therapy. The cationic liposome is a non virus carrier which is widely used to deliver siRNA. It is non-toxic, can be degraded automatically and has no immunogenicity. It can be synthesized and magnified in large quantities. In recent years, the advantages of production have been paid much attention by researchers.
The research group has been working on the preparation and activity of cationic liposomes delivering siRNA in the early period. The previous study found that a PEG immunized freeze-dried liposome (Lyophilized PEGylated Immunoliposomes, LPIL) was prepared by the treatment of cationic liposomes, such as polyethylene glycol (PEG), HER2 antibody modification and freeze drying. When the content of PEG is 2.5 mol%, this liposome can effectively deliver siRNA specific delivery to HER2 breast cancer cells and silence the expression of related genes..PEG can significantly increase the stability of liposomes in plasma and prolong the half-life of the drug. When the PEG content is more than 8 mol%, the surface of liposomes with a particle size of 100 nm is brushed This brush conformation can completely cover the surface of the liposome and provide more comprehensive protection for the liposomes to avoid the phagocytosis of the reticuloendothelial system. However, the PEG content of LPIL is low and the application in the body is limited. And LPIL can not introduce a higher content of PEG, because the higher content of PEG will destroy the physical stability of LPIL and will greatly reduce the siRNA. Therefore, we urgently need to develop a cationic liposome with high PEG content, high siRNA entrapment efficiency and good targeting.
LPD (liposome-polycation-DNA, liposome polycationic -DNA complex) is a new type of cationic liposome that delivers siRNA. Its structure is PEG (polyethylene glycol) encapsulated protamine, siRNA/DNA complex and cationic liposome. Unlike the traditional cationic liposome, DNA is compressed by the protamine of protamine in the action of protamine. The three formed a tight negative nucleus and formed a stable LPD through the self assembly process after mixing with cationic liposomes. It is particularly important that LPD be inserted into PEG after the insertion of a high content PEG on its surface, which not only ensures the high encapsulation efficiency of siRNA, but also increases the stability of the liposomes, and is proved in the body. SiRNA can be effectively delivered to tumor cells. However, the antibody modified LPD has not yet systematically explored its various nanoscale characterization and in vivo and in vivo activity. This study is based on the early PEG immunization of cationic liposomes and LPD. Through a series of prescription screening, a EGFR targeted immunliposome with high PEG content was first optimized. TLPD-FCC, and its various nano characterization and in vivo and in vitro activities have been studied and discussed in depth.
First, we mixed the DOTAP/Chol cationic liposomes with protamine, calf thymus DNA, and siRNA to get Naked LPD (non PEG liposome), and then prepare the EGFR targeting immunliposome TLPD by PEG and introducing the antibody (Anti-EGFR mAb or Fab '). Against the effect of the antibody on the size of the liposomes and the potential of the liposome, the antagonism class The model, connection mode and input were optimized. The results showed that the antibody type was Anti-EGFR Fab ', and the liposome TLPD-FC was prepared when the antibody was connected by the traditional connection. The average particle size was between 150 nm~160 nm and the zeta potential was about 10 mV, which laid the foundation for the follow-up experimental research.
Then, the SDS-PAGE experiment confirmed that the antibody did connect to the liposomes, and also examined Naked-LPD, NTLPD (PEG non targeting liposome), TLPD-FC to siRNA binding capacity, the encapsulation efficiency of siRNA and the efficiency of gene silencing in vitro. The results of gel block experiment showed that Naked-LPD, NTLPD, TLPD-FC had strong binding ability to siRNA. The method of ultra filtration centrifugation confirmed that the encapsulation efficiency of siRNA was as high as 90%. The above experiments confirmed that the above samples had a strong encapsulation ability of siRNA, and PEG or antibody modification had little effect on the siRNA encapsulation efficiency. In vitro gene silencing efficiency examined the TLPD-FC (including TLPD-FCA, TLPD-FCB, TLPD-FCC, TLPD-FCD) in MDA-MB-231 (including TLPD-FCA, TLPD-FCB, TLPD-FCC, TLPD-FCD) in MDA-MB-231. The efficiency of gene silencing in cells shows that TLPD-FCC has the highest gene silencing efficiency.
Finally, the related properties and in vivo and in vivo activity of TLPD-FCC were deeply studied and discussed. The morphology and size distribution of TLPD-FCC and NTLPD were observed by transmission electron microscopy, and there was no obvious difference in the morphology and size distribution. It showed that the antibody connection had little effect on the structure of liposomes. The agarose gel electrophoresis test confirmed the siR The serum stability of NA was good under the protection of TLPD-FCC or NTLPD. The serum stability of liposomes was concluded by the dynamic light scattering experiment. Compared with Naked LPD, NTLPD or TLPD-FCC was not easy to interact with BSA under the protection of PEG. The results of gene transfection efficiency and gene silence efficiency in vitro showed that TLPD-FCC was compared with NTLPD. High specific transfection efficiency and gene silencing activity. Subsequently, the immunofluorescent labeling experiment confirmed the EGFR expression level of the tumor cells in the body through the establishment of the MDA-MB-231 breast cancer tumor model. In vivo distribution experiments also demonstrated that TLPD-FCC was highly aggregated with the time in the tumor site through the receptor mediated endocytosis. The peak value was reached, and the laser confocal results showed that TLPD-FCC showed the targeting specificity and binding ability and endocytosis efficiency of tumor cells higher than that of NTLPD. Finally, the results of gene silencing efficiency in the body showed that TLPD-FCC had higher gene silencing efficiency than NTLPD and had specific gene silencing activity.
The TLPD-FCC obtained in this study can effectively deliver siRNA to EGFR in breast cancer cells, and has good gene silencing efficiency in vitro and in vivo. It may be used as a gene carrier for the treatment of high expression of EGFR breast cancer.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
本文編號：2123047
[Abstract]:Gene therapy is an ideal treatment for the treatment of hereditary diseases or acquired acquired diseases, especially the treatment of.RNAi (RNA interfering, RNA interference), as an efficient sequence specific gene silencing technique in the field of malignant tumor gene therapy, set off a hot research upsurge, of which, siRNA (small in). Terfering RNA, small interference RNA) is an effector in the RNAi path. It is a short segment double chain RNA of 21~23 BP, which can specifically degrade mRNA of the homologous sequence, inhibit the expression of specific tumor related genes, and thus inhibit the growth, invasion and metastasis of tumor. But, siRNA can not enter the tumor fine without the help of the carrier. In the cell, the carrier is the most important problem that restricts the siRNA gene therapy. Therefore, the research on the delivery carrier of siRNA is a hot issue in the research of tumor gene therapy. The cationic liposome is a non virus carrier which is widely used to deliver siRNA. It is non-toxic, can be degraded automatically and has no immunogenicity. It can be synthesized and magnified in large quantities. In recent years, the advantages of production have been paid much attention by researchers.
The research group has been working on the preparation and activity of cationic liposomes delivering siRNA in the early period. The previous study found that a PEG immunized freeze-dried liposome (Lyophilized PEGylated Immunoliposomes, LPIL) was prepared by the treatment of cationic liposomes, such as polyethylene glycol (PEG), HER2 antibody modification and freeze drying. When the content of PEG is 2.5 mol%, this liposome can effectively deliver siRNA specific delivery to HER2 breast cancer cells and silence the expression of related genes..PEG can significantly increase the stability of liposomes in plasma and prolong the half-life of the drug. When the PEG content is more than 8 mol%, the surface of liposomes with a particle size of 100 nm is brushed This brush conformation can completely cover the surface of the liposome and provide more comprehensive protection for the liposomes to avoid the phagocytosis of the reticuloendothelial system. However, the PEG content of LPIL is low and the application in the body is limited. And LPIL can not introduce a higher content of PEG, because the higher content of PEG will destroy the physical stability of LPIL and will greatly reduce the siRNA. Therefore, we urgently need to develop a cationic liposome with high PEG content, high siRNA entrapment efficiency and good targeting.
LPD (liposome-polycation-DNA, liposome polycationic -DNA complex) is a new type of cationic liposome that delivers siRNA. Its structure is PEG (polyethylene glycol) encapsulated protamine, siRNA/DNA complex and cationic liposome. Unlike the traditional cationic liposome, DNA is compressed by the protamine of protamine in the action of protamine. The three formed a tight negative nucleus and formed a stable LPD through the self assembly process after mixing with cationic liposomes. It is particularly important that LPD be inserted into PEG after the insertion of a high content PEG on its surface, which not only ensures the high encapsulation efficiency of siRNA, but also increases the stability of the liposomes, and is proved in the body. SiRNA can be effectively delivered to tumor cells. However, the antibody modified LPD has not yet systematically explored its various nanoscale characterization and in vivo and in vivo activity. This study is based on the early PEG immunization of cationic liposomes and LPD. Through a series of prescription screening, a EGFR targeted immunliposome with high PEG content was first optimized. TLPD-FCC, and its various nano characterization and in vivo and in vitro activities have been studied and discussed in depth.
First, we mixed the DOTAP/Chol cationic liposomes with protamine, calf thymus DNA, and siRNA to get Naked LPD (non PEG liposome), and then prepare the EGFR targeting immunliposome TLPD by PEG and introducing the antibody (Anti-EGFR mAb or Fab '). Against the effect of the antibody on the size of the liposomes and the potential of the liposome, the antagonism class The model, connection mode and input were optimized. The results showed that the antibody type was Anti-EGFR Fab ', and the liposome TLPD-FC was prepared when the antibody was connected by the traditional connection. The average particle size was between 150 nm~160 nm and the zeta potential was about 10 mV, which laid the foundation for the follow-up experimental research.
Then, the SDS-PAGE experiment confirmed that the antibody did connect to the liposomes, and also examined Naked-LPD, NTLPD (PEG non targeting liposome), TLPD-FC to siRNA binding capacity, the encapsulation efficiency of siRNA and the efficiency of gene silencing in vitro. The results of gel block experiment showed that Naked-LPD, NTLPD, TLPD-FC had strong binding ability to siRNA. The method of ultra filtration centrifugation confirmed that the encapsulation efficiency of siRNA was as high as 90%. The above experiments confirmed that the above samples had a strong encapsulation ability of siRNA, and PEG or antibody modification had little effect on the siRNA encapsulation efficiency. In vitro gene silencing efficiency examined the TLPD-FC (including TLPD-FCA, TLPD-FCB, TLPD-FCC, TLPD-FCD) in MDA-MB-231 (including TLPD-FCA, TLPD-FCB, TLPD-FCC, TLPD-FCD) in MDA-MB-231. The efficiency of gene silencing in cells shows that TLPD-FCC has the highest gene silencing efficiency.
Finally, the related properties and in vivo and in vivo activity of TLPD-FCC were deeply studied and discussed. The morphology and size distribution of TLPD-FCC and NTLPD were observed by transmission electron microscopy, and there was no obvious difference in the morphology and size distribution. It showed that the antibody connection had little effect on the structure of liposomes. The agarose gel electrophoresis test confirmed the siR The serum stability of NA was good under the protection of TLPD-FCC or NTLPD. The serum stability of liposomes was concluded by the dynamic light scattering experiment. Compared with Naked LPD, NTLPD or TLPD-FCC was not easy to interact with BSA under the protection of PEG. The results of gene transfection efficiency and gene silence efficiency in vitro showed that TLPD-FCC was compared with NTLPD. High specific transfection efficiency and gene silencing activity. Subsequently, the immunofluorescent labeling experiment confirmed the EGFR expression level of the tumor cells in the body through the establishment of the MDA-MB-231 breast cancer tumor model. In vivo distribution experiments also demonstrated that TLPD-FCC was highly aggregated with the time in the tumor site through the receptor mediated endocytosis. The peak value was reached, and the laser confocal results showed that TLPD-FCC showed the targeting specificity and binding ability and endocytosis efficiency of tumor cells higher than that of NTLPD. Finally, the results of gene silencing efficiency in the body showed that TLPD-FCC had higher gene silencing efficiency than NTLPD and had specific gene silencing activity.
The TLPD-FCC obtained in this study can effectively deliver siRNA to EGFR in breast cancer cells, and has good gene silencing efficiency in vitro and in vivo. It may be used as a gene carrier for the treatment of high expression of EGFR breast cancer.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
【共引文獻】
相關(guān)期刊論文 前5條
1 徐海明;蔣稼歡;;微流控技術(shù)在細胞轉(zhuǎn)染中的應(yīng)用[J];生物工程學(xué)報;2011年10期
2 趙曉軍;謝忠明;;基因治療中基因轉(zhuǎn)移載體的研究進展[J];微循環(huán)學(xué)雜志;2010年04期
3 何曉紅;曹少先;茆達干;楊利國;;影響生長抑素重組質(zhì)粒pEGS/2SS轉(zhuǎn)染HeLa細胞效果的因素研究[J];畜牧獸醫(yī)學(xué)報;2010年03期
4 別彩群;韓利民;王啟章;范紅梅;朱浩峰;梁旭競;楊冬華;;抗肝癌免疫納米顆粒的制備及其對肝癌細胞增殖的影響[J];世界華人消化雜志;2010年13期
5 劉薇;陳建明;;陽離子脂質(zhì)在基因傳遞中構(gòu)效關(guān)系的研究進展[J];中國新藥雜志;2011年20期
相關(guān)碩士學(xué)位論文 前4條
1 周霞;大鼠TGF-β_3基因的克隆及其對肝星狀細胞TGF-β_1和I型膠原合成的影響[D];華中科技大學(xué);2007年
2 趙陽;明膠—硅氧烷納米顆粒作為基因載體的應(yīng)用[D];廈門大學(xué);2008年
3 馬慧潔;SV40T抗原基因的穩(wěn)定轉(zhuǎn)染及對人胃癌細胞SGC7901的生物學(xué)效應(yīng)[D];鄭州大學(xué);2008年
4 王貝;殼寡糖作為基因遞送載體的實驗研究[D];湖南大學(xué);2010年
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