本文選題：脂質(zhì)基因載體 + PEG修飾��； 參考：《上海交通大學(xué)》2012年博士論文

【摘要】：在過去的二十多年間，基因治療已經(jīng)在很多疾病治療領(lǐng)域?qū)⒀芯繌呐R床前推向了臨床，這其中既有單基因疾病比如囊腫性纖維化和杜氏肌營養(yǎng)不良癥的治療，也包括更復(fù)雜的疾病比如癌癥和心血管疾病的治療。迄今為止最成功的案例當(dāng)屬重癥聯(lián)合免疫缺陷的基因治療，不過在許多其他領(lǐng)域基因治療的進(jìn)展也非常引人矚目。雖然在研究的推進(jìn)過程中發(fā)現(xiàn)有的疾�。ū热缒夷[性纖維化）的基因治療比預(yù)想要困難、見效要慢，使得工業(yè)界和學(xué)術(shù)界對這些疾病基因治療的關(guān)注熱度在過去十多年間有所下降，但是基因治療對于不宜手術(shù)的和對傳統(tǒng)療法缺乏響應(yīng)的疾病仍具有無可替代的優(yōu)勢，結(jié)合在多種疾病的臨床治療中取得的成績，，仍然表明了基因治療是未來醫(yī)學(xué)的希望之一。 載體是基因治療的重要組成部分，是影響治療結(jié)果的重要因素。因?yàn)榛蛩幬锉旧聿荒苓M(jìn)入細(xì)胞，在體內(nèi)環(huán)境中又極易被降解，因此起保護(hù)和攜載作用的載體會(huì)直接影響到基因治療的效果�？v觀進(jìn)入臨床試驗(yàn)的基因藥物多是選擇使用病毒類基因載體，這是由于病毒類載體具有較高的基因輸送效率。但是許多這類臨床實(shí)驗(yàn)止步于I期或II期，很大程度上是因?yàn)椴《绢愝d體的免疫原性和毒性較高。即使有些臨床實(shí)驗(yàn)采用了ex vivo的策略，即在體外用病毒載體轉(zhuǎn)染細(xì)胞，再將細(xì)胞通過手術(shù)途徑植入病灶部位的方法避開人體免疫系統(tǒng)的攻擊，病人仍然需要周期性的動(dòng)手術(shù)取出老細(xì)胞植入新細(xì)胞，這不但增加了手術(shù)中感染的風(fēng)險(xiǎn)，也不利于病人接受。而非病毒類載體具有低毒性，低免疫原性，低致癌性的優(yōu)點(diǎn)，大大提高了使用安全性。除此之外，非病毒類載體對攜載基因的大小沒有限制，可以重復(fù)給藥，質(zhì)量控制簡單，制備可重復(fù)性高，這些優(yōu)勢都使得非病毒類載體成為未來最終實(shí)現(xiàn)基因治療的可能方式。并且可用于系統(tǒng)給藥的非病毒類載體，由于注射方式簡單、便捷、微創(chuàng)，最終將成為治療趨勢發(fā)展的方向。 非病毒類載體成功實(shí)現(xiàn)體內(nèi)基因治療的前提包括：首先，載體在循環(huán)系統(tǒng)中保持穩(wěn)定以提高在病灶部位的基因藥物濃度；其次，載體在靶細(xì)胞內(nèi)及時(shí)高效地將基因藥物釋放到細(xì)胞質(zhì)中以提高轉(zhuǎn)染的效率。本論文按照這兩個(gè)前提，首先采用高于傳統(tǒng)修飾量的PEG脂質(zhì)對基因載體Lipid-mu peptide-DNA（LMD）進(jìn)行表面修飾，提高了載體在腫瘤部位的聚集程度；其次使用質(zhì)子泵抑制劑類藥物奧美拉唑抑制細(xì)胞中內(nèi)涵體或溶酶體的酸化過程，提高了載體的體外和體內(nèi)基因轉(zhuǎn)染效率。具體內(nèi)容和主要結(jié)果總結(jié)如下。 和普通脂質(zhì)DNA復(fù)合物的制備過程不同，LMD的制備是先將帶負(fù)電的基因快速加入到帶正電的多肽mu中，通過正負(fù)電荷相互作用形成帶有負(fù)電的mu-DNA（MD）粒子，再將MD粒子加入到含有陽離子脂質(zhì)的脂質(zhì)體中，再次通過正負(fù)電荷相互作用形成凈電荷為正電的LMD粒子。整個(gè)制備過程可以描述為mu，DNA和脂質(zhì)體的自發(fā)組裝，制備耗時(shí)短，利于重復(fù)。通過對粒徑等物理化學(xué)性質(zhì)和體外轉(zhuǎn)染實(shí)驗(yàn)的優(yōu)化，我們確定了LMD三種組分間的最佳比例DNA:mu:cationic lipid是1mg:0.6mg:12μmol。在這個(gè)比例下得到MD粒徑為102±12nm，zeta電位為-33±1mV；LMD粒徑為140±15nm，zeta電位為30±0.3mV。之后我們使用原子力顯微鏡觀察到LMD的形貌是球形或接近球形的粒子，大小比較均一。結(jié)合Miller教授實(shí)驗(yàn)室的冰凍蝕刻電鏡圖像，可知LMD具有MD粒子在核心，外面包裹脂質(zhì)雙層的結(jié)構(gòu)。和傳統(tǒng)脂質(zhì)復(fù)合物相比，這種結(jié)構(gòu)更加穩(wěn)定，制備可重復(fù)性更高，并且可以在其表面修飾高于傳統(tǒng)修飾量（10mol%）的PEG脂質(zhì)。 由于PEG脂質(zhì)頭部的多羥基結(jié)構(gòu)具有良好的親水性，PEG聚合物長鏈又可以起到空間位阻的作用，故可以降低被修飾粒子和血液循環(huán)中蛋白間相互作用引起的調(diào)理作用，減少粒子和帶負(fù)電蛋白之間聚集而形成大顆粒的程度，增加載體粒子在體內(nèi)穩(wěn)定性，延長循環(huán)時(shí)間，因此PEG修飾的載體也稱為長循環(huán)載體。我們通過后期孵育法制備了可用于系統(tǒng)給藥的長循環(huán)PEG-LMD。經(jīng)過高于傳統(tǒng)PEG修飾量（15-25mol%）修飾的LMD的zeta電位由30mV變成接近0，粒徑則比修飾前略有增大，為170±30nm。 由于實(shí)體瘤的高通透性和滯留效應(yīng)（Enhanced Permeability andRetention，EPR效應(yīng)）長循環(huán)載體可以被動(dòng)靶向到皮下移植瘤裸鼠的腫瘤部位。較高PEG表面修飾的LMD粒子可通過EPR效應(yīng)更多地聚集在腫瘤部位，我們通過小動(dòng)物活體成像、冰凍切片、免疫組化和實(shí)時(shí)PCR實(shí)驗(yàn)分別證實(shí)了此結(jié)論。使用近紅外熒光染料Cy5.5標(biāo)記LMD的脂質(zhì)成分，我們使用小動(dòng)物成像儀對一系列不同PEG表面密度的LMD粒子（0，10，15，23，25，30mol%）在不同時(shí)間點(diǎn)于腫瘤處的分布進(jìn)行了直觀的觀測：尾靜脈注射后，熒光信號很快聚集在腫瘤部位，在較高PEG修飾密度下，比如15%-25%范圍內(nèi)，Cy5.5-PEG-LMD在腫瘤中有較強(qiáng)的熒光信號，在注射5天后，依然可以在腫瘤部位觀察到明顯的熒光信號，并且腫瘤部位的熒光強(qiáng)度持續(xù)高于其他組織。我們進(jìn)一步從熒光圖片上提取出相關(guān)組織和腫瘤的熒光強(qiáng)度，用組織面積和全身熒光總強(qiáng)度進(jìn)行歸一化處理，使用非房室模型對數(shù)據(jù)進(jìn)行分析，得到了一致的結(jié)論。我們通過切片染色的方法用激光共聚焦顯微鏡觀察分別標(biāo)記了基因物質(zhì)和脂質(zhì)成分的LMD在腫瘤的分布，同樣觀察到含有23mol%PEG的LMD比只有2mol%或10mol%PEG的LMD能夠更多地分布在腫瘤部位。并且23%PEG-LMD在不同制劑批次間和不同小鼠體內(nèi)都表現(xiàn)出很強(qiáng)的瘤內(nèi)熒光信號。最后我們通過實(shí)時(shí)PCR對不同PEG修飾密度的LMD在腫瘤部位的分布進(jìn)行了定量分析，得到相同結(jié)論：不同PEG修飾密度的LMD腫瘤含量為25%23%15%30%10%。 在優(yōu)化PEG-LMD體內(nèi)腫瘤分布后，我們接著考察了載體的基因轉(zhuǎn)染能力。我們使用Luciferase質(zhì)粒DNA作為報(bào)告基因，多輪體外和體內(nèi)基因轉(zhuǎn)染實(shí)驗(yàn)結(jié)果顯示經(jīng)過PEG修飾的LMD粒子無法進(jìn)行有效轉(zhuǎn)染。我們推測造成PEG-LMD轉(zhuǎn)染效率低下的原因可能是PEG脂質(zhì)阻礙了載體從內(nèi)涵體中逃逸，隨著內(nèi)涵體pH值降低以及和溶酶體融合，載體粒子中的基因物質(zhì)最終被酶解而喪失轉(zhuǎn)染能力。為了增加載體從內(nèi)涵體中逃逸的程度，我們首次將質(zhì)子泵抑制劑類藥物奧美拉唑和基因輸送聯(lián)合應(yīng)用�；谫|(zhì)子泵抑制劑可以阻礙內(nèi)涵體和溶酶體中pH值降低，我們假設(shè)這一特性可以增加載體粒子從內(nèi)涵體逃逸的程度。實(shí)驗(yàn)證明有質(zhì)子泵抑制劑存在下，移去熒光標(biāo)記的23%PEG-LMD6天后依然可以觀察到細(xì)胞內(nèi)的熒光，而沒有質(zhì)子泵抑制劑存在下的細(xì)胞觀察不到明顯熒光信號，并且熒光強(qiáng)度和質(zhì)子泵抑制劑的濃度成正相關(guān)關(guān)系。體外轉(zhuǎn)染實(shí)驗(yàn)表明0.1mg/ml奧美拉唑可將23%PEG-LMD的H1299細(xì)胞轉(zhuǎn)染結(jié)果提高9倍。當(dāng)使用碳鏈較短的PEG脂質(zhì)DPPE-PEG時(shí)，0.1mg/ml奧美拉唑可將23%PEG-LMD的H1299細(xì)胞轉(zhuǎn)染結(jié)果提高34倍。體內(nèi)轉(zhuǎn)染實(shí)驗(yàn)中150mg/kg奧美拉唑可將23%PEG-LMD對H1299皮下移植瘤的轉(zhuǎn)染效率提高3倍多。實(shí)驗(yàn)結(jié)果顯示奧美拉唑可以通過抑制內(nèi)涵體和溶酶體pH值降低，增加PEG化脂質(zhì)載體LMD的體外和體內(nèi)基因轉(zhuǎn)染效率。
[Abstract]:In the past more than 20 years, gene therapy has been pushed into clinical trials in many areas of disease treatment, including the treatment of single gene diseases such as cystic fibrosis and dystrophy, including more complex diseases such as cancer and heart disease. The most successful cases to date. The genetic treatment of severe combined immunodeficiency, but the progress of gene therapy in many other fields is also very attractive. Although the progress of research has found that some diseases, such as cystic fibrosis, are more difficult than expected and are slower to enable industry and academia to treat these diseases by gene therapy. The heat of concern has declined over the past more than 10 years, but gene therapy still has an irreplaceable advantage for Unoperable and unresponsive diseases of traditional therapies. The achievements in the clinical treatment of various diseases still indicate that gene therapy is one of the hopes of future medicine.
The carrier is an important component of gene therapy, which is an important factor affecting the outcome of the treatment. Because the gene drug itself cannot enter the cell and is easily degraded in the body environment, the carrier of protective and carrying action will directly affect the effect of gene therapy. This is due to the high gene delivery efficiency of viral vectors. However, many of these clinical trials stop at stage I or II, largely due to the high immunogenicity and toxicity of viral vectors. Even some clinical trials have adopted the strategy of ex vivo, that is, transfection of cells with viral vectors in vitro, and then To avoid the attack of the body's immune system by surgical approaches, the patients still need periodic operations to remove the old cells into the new cells, which not only increases the risk of infection in the operation, but also is not conducive to the patient's acceptance, but the non viral vector has the advantages of low toxicity, low immunogenicity and low carcinogenicity. In addition, the non viral vectors have no limitation on the size of the carrying genes, can be repeated to the drug, the quality control is simple, and the preparation is high repeatability. These advantages make the non viral vector a possible way to eventually realize the gene therapy in the future. And it can be used for the non viral vector of the system for drug delivery. Because the injection method is simple, convenient and minimally invasive, it will eventually become the trend of treatment trend.
The precondition of the successful implementation of gene therapy in the body of non viral vectors includes: first, the carrier is stable in the circulatory system to improve the gene drug concentration at the site of the focus. Secondly, the carrier is released into the cytoplasm in time and efficiently in the target cells to improve the efficiency of transfection. First of all, this paper is based on these two prerequisites. The surface modification of the gene carrier Lipid-mu peptide-DNA (LMD) was carried out with the PEG lipid higher than the traditional modifier to improve the concentration of the carrier in the tumor site. Secondly, the acidification process of the endosome or lysosome in the cells was inhibited by omeprazole, the proton pump inhibitor drug, to improve the gene transfection efficiency in vitro and in vivo. The specific content and main results are summarized as follows.
The preparation process of the DNA complex is different from that of the common lipid complex. The preparation of LMD is to quickly add negative electricity to the positive peptide mu, to form a negative mu-DNA (MD) particle by the positive and negative charge interaction, and then add MD particles to the liposomes containing the cationic lipid, and then form a positive and negative charge interaction. The entire preparation process can be described as the spontaneous assembly of mu, DNA and liposomes, and the preparation of the spontaneous assembly of DNA and liposomes is short and beneficial to repetition. By optimizing the physical and chemical properties of the particle size and in vitro transfection experiments, we have determined that the optimum proportion of DNA: mu:cationic lipid between the three components of the LMD is 1mg:0.6mg:12 u mol. in this proportion. When the particle size of MD is 102 + 12NM, zeta potential is -33 + 1mV, LMD particle diameter is 140 + 15nm, and zeta potential is 30 + 0.3mV., we use atomic force microscope to observe that the morphology of LMD is spherical or close to spherical particles, and the size is uniform. The structure is coated with lipid bilayer. Compared with the traditional lipid complex, this structure is more stable, more reproducible, and can modify the PEG lipid on its surface, which is higher than the traditional modification (10mol%).
Because the polyhydroxy structure of the PEG lipid head has a good hydrophilic property, the long chain of PEG can also play the role of space hindrance, so it can reduce the conditioning effect caused by the interprotein interaction between the modified particles and the blood circulation, and reduce the degree of particle and the accumulation of negative proteins to form large particles and increase the carrier particle. The PEG modified carrier is also known as a long cycle carrier in the body, so we have prepared a long cycle PEG-LMD. that can be used for system administration by the later incubation method. The zeta potential of LMD, which is higher than the traditional PEG modification (15-25mol%), is changed from 30mV to nearly 0, and the particle size is slightly larger than that before the modification, which is 170 + 30nm..
Due to the high permeability and retention effect of solid tumor (Enhanced Permeability andRetention, EPR effect) long circulation vector can be passively targeted to the tumor site of nude mice of subcutaneous transplanted tumor. Higher PEG surface modified LMD particles can accumulate more at the tumor site through the EPR effect. We use small animals in vivo imaging, frozen section, immunization This conclusion was confirmed by the histochemical and real-time PCR experiments. Using the near infrared fluorescent dye Cy5.5 labeled LMD lipid components, we used the small animal imager to observe the distribution of a series of LMD particles (0,10,15,23,25,30mol%) with different PEG surface density (0,10,15,23,25,30mol%) at different time points at the tumor. After the injection of the tail vein, the fluorescent letter was used. The number is quickly gathered at the tumor site. At the high PEG modification density, such as the 15%-25% range, Cy5.5-PEG-LMD has strong fluorescence signal in the tumor. After 5 days of the injection, the obvious fluorescence signal can still be observed at the tumor site, and the fluorescence intensity of the tumor site is higher than that of the other tissues. The fluorescence intensity of the related tissues and tumors was extracted, the tissue area and total body fluorescence intensity were normalized, and the data were analyzed with the non atrioventricular model, and a consistent conclusion was obtained. We used laser confocal microscopy to observe the LMD in the tumor by laser confocal microscopy. The distribution of 23mol%PEG was also observed to be more distributed at the tumor site than that of LMD with only 2mol% or 10mol%PEG. And 23%PEG-LMD showed strong intratumoral fluorescence signals between different batches of different preparations and in different mice. Finally, the distribution of LMD at the tumor site by real time PCR on different PEG modification densities was distributed. Quantitative analysis showed that the tumor size of LMD with different PEG density was 25%23%15%30%10%.
After optimizing the tumor distribution in PEG-LMD, we then examined the gene transfection capacity of the carrier. We used the Luciferase plasmid DNA as the reporter gene, and the results of gene transfection experiments in vitro and in vivo showed that the PEG modified LMD particles could not be transfected effectively. The reasons for the low efficiency of PEG-LMD transfection were possible. It is the PEG lipid that prevents the carrier from escaping from the endosome, and with the decrease of the endosome pH and the fusion of the lysosomes, the gene material in the carrier particles eventually loses the transfection capacity. In order to increase the extent of the escape of the carrier from the endosome, the proton pump inhibitor drugs omeprazole and gene delivery are combined for the first time. Proton pump inhibitors can inhibit the reduction of pH values in the endosomes and lysosomes. We assume that this property can increase the degree of escape of the carrier particles from the endosome. Experiments show that the proton pump inhibitors exist in the presence of 23%PEG-LMD6 days after removing the fluorescent labeling, and there are no proton pump inhibitors. No obvious fluorescent signal was observed in the cells below, and the fluorescence intensity was positively correlated with the concentration of proton pump inhibitors. In vitro transfection experiments showed that 0.1mg/ml omeprazole could increase the transfection result of 23%PEG-LMD H1299 cells by 9 times. When the PEG lipid DPPE-PEG of a shorter carbon chain was used, the 0.1mg/ml omeprazole could be the H1299 of 23%PEG-LMD. The results of cell transfection were increased by 34 times. 150mg/kg omeprazole could increase the transfection efficiency of 23%PEG-LMD to H1299 subcutaneous tumor by 3 times. The results showed that omeprazole could decrease the pH value of endosome and lysosome, and increase the gene transfection efficiency of PEG liposome LMD in vivo and in vivo.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R3416

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