磁性Fe_3O_4纳米微粒介导TRAIL基因治疗涎腺腺样囊性癌的研究
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摘要
腺样囊性癌（Adenoid cystic carcinoma，ACC）是最常见的涎腺恶性肿瘤之一，约占涎腺上皮肿瘤的~10%。其发生率在口腔颌面部肿瘤中占第二位。与其他涎腺恶性肿瘤相比，生长速度较慢，但预后相对较差。然而，现有的治疗方法包括手术、放疗、化疗及联合治疗均不能提高患者的生存率。为此，国内外的学者正试图寻找新的更有效的治疗方法。目前为止，恶性肿瘤仍然是导致人类死亡的主要问题，而他的治疗一直以来困扰人们。过去的几十年来，随着基因治疗的发展，恶性肿瘤的转基因治疗也开始兴起。转基因治疗是指将外源性基因导入体细胞以纠正遗传或后天获得的基因缺陷，经过20余年的探索与研究，转基因治疗在遗传性疾病、代谢性疾病、恶性肿瘤的治疗领域己经取得很大进步，个别己应用于临床。这一治疗方法具有巨大的潜力，但是安全有效的应用于人体之前，仍有很多问题需要解决，例如特异性、转染效率、毒副作用等等。在目前的研究中，我们努力开发一种新的纳米微粒系统达到特异的、高效的转染效率、低毒副作用的癌症转基因治疗。
肿瘤坏死因子相关凋亡诱导配体TRAIL属于肿瘤坏死因子超家族。TRAIL可以诱导很多肿瘤细胞系凋亡。TRAIL的杀伤作用是肿瘤特异性的，对正常细胞及组织几乎没有副作用。虽然对于这种肿瘤特异性杀伤性作用的机制几乎不知道，但是TRAIL仍被认为是肿瘤治疗可能的有效的治疗肿瘤的有效基因，能为肿瘤治疗提供新的思路。人端粒酶逆转录酶(hTERT)在85-90%的恶性肿瘤中高表达，而在正常体细胞中几乎没有表达。所以hTERT被认为是肿瘤转基因治疗的特异性启动子。许多研究都将hTERT启动子使抗肿瘤基因在肿瘤细胞中靶向表达，对正常细胞没有杀伤作用，减少了其副作用。我们之前的研究表明，腺病毒载体编码TRAIL基因，并由hTERT启动子介导在体内可以有效杀伤ACC。本实验中我们设计使用质粒载体pACTERT-TRAIL。
近年来，纳米技术飞速发展，并在很多医学领域有广泛的应用，尤其是多功能的纳米微粒的医学应用。纳米或者微米颗粒的表面可以与许多分子结合，例如DNA分子。磁性纳米微粒是一类特殊的纳米微粒，已经被用于药物输送、快速生物分离等方面。一些以前的研究表明DNA分子能通过共价键与磁性纳米微粒复合，在外加磁场作用下可以提高转染效率。
在国家自然科学基金的资助下（30672338、30740420551、30830108），我们将转基因治疗与纳米技术联合开发了一种新的治疗方法。合成了以人端粒酶逆转录酶为启动子，表达肿瘤坏死因子相关凋亡诱导配体TRAIL的质粒载体pACTERT-TRAIL。此外合成了带正电的聚合物PEI修饰Fe_3O_4纳米微粒，使其与带负电的质粒载体复合，形成了Fe_3O_4-PEI-质粒复合物(FPP)。我们在碱性共沉淀方法里面加入了分枝状聚合物PEI，合成了PEI修饰Fe_3O_4纳米微粒作为载体。纳米微粒表面的胺基可以与质粒DNA的磷酸根通过静电作用复合形成含DNA的FPP。非病毒载体系统更加安全具有更小的副作用。它可以局部靶向输送DNA。溶酶体内聚合物PEI通过质子海绵效应保护DNA不被降解。并且FPP带正电可以与带负电的细胞膜复合，从而更快的进入细胞。研究表明：与带负电和中性的粒子相比，带正电的粒子可以与细胞结合从而更快的被细胞吞噬。
PEI修饰的Fe_3O_4磁性纳米微粒组在Nd-Fe-B外加磁场作用下，将pACTERT-TRAIL转染SACC-83细胞，通过MTT、流式细胞仪等技术，观察其对SACC-83增殖和凋亡作用；体内以腺样囊性癌裸鼠移植瘤模型为研究对象，外加磁场作用下磁转染pACTERT-TRAIL，测量肿瘤的大小、检测目的基因的表达以及观察裸鼠脏器形态学和血液学等改变，探讨pACTERT-TRAIL对实验动物体内腺样囊性癌的治疗作用。结果发现，体外转染实验中，PEI修饰的Fe_3O_4磁性纳米微粒组在Nd-Fe-B外加磁场作用下，获得了更高的转染效率。MTT实验及Annexin V-FITC/PI双染测定SACC-83细胞的凋亡比率结果表明，PEI修饰的Fe_3O_4磁性纳米微粒组在Nd-Fe-B外加磁场作用下更加有效的杀伤靶细胞。体内实验中，磁性纳米载体组在外加磁场作用下，能显著抑制腺样囊性癌裸鼠移植瘤生长，并且荷瘤裸鼠的心脏、肝脏、肾脏、肺脏，未见明显的形态学改变，全血中红细胞、白细胞、血小板数目，血红蛋白含量也未见明显变化。
人TRAIL具有选择性的肿瘤特异性杀伤活性，而对大多数正常细胞和组织几乎没有杀伤作用。然而以往的研究结果表明，高浓度的TRAIL在体外可诱发肝细胞和人脑细胞的毒性。TRAIL的毒性还包括对正常组织具有潜在的缺血性和出血性反应。为了尽量减少对TRAIL潜在的副作用，并限制在正常细胞中的表达，我们选择了hTERT启动子作为恶性肿瘤特异性启动子。我们以前的研究曾显示，hTERT启动子选择性介导的TRAIL在SACC-83中表达并有效杀伤了癌细胞。这些细胞的特异性杀伤肿瘤细胞作用中，我们使用的PEI修饰的氧化铁纳米粒子输送质粒进入靶细胞，通过在外加的Nd-Fe-B磁场作用下，可以将纳米质粒复合微粒进一步限制在肿瘤细胞。因此，PEI修饰的Fe_3O_4磁性纳米微粒在Nd-Fe-B磁场作用下与腺病毒载体相比更加简单，具体，高效，低毒性的转染方法。
总之，这项研究表明，我们能够创建一个有效的PEI修饰的Fe_3O_4磁性纳米微粒，它可以连接质粒DNA，在磁场作用下可以有效地转染ACC的肿瘤细胞SACC-83，体内外均取得了较好的抑制肿瘤的作用。PEI修饰的Fe_3O_4磁性纳米微粒治疗具有很大的临床应用潜力。
ACC is one of the most common malignant neoplasms in the human salivary gland，representing~10%of salivary gland tumors. Adenoid cystic carcinoma is the second mostcommon malignant tumor in the salivary gland.The ACC grows relatively slowly and has a poorprognosis compared to other malignant tumors in salivary glands. However， the currenttreatments for ACC，including surgery， radiation therapy， chemotherapy and combinationsthereof，still could not improve overall mortality rates. To efficiently and successfully treatACC， new treatments are needed. To date， cancer is still one of major lethal diseases inhuman for which treatment can be problematic. Over decade ago， cancer gene therapy startedto develop with the gene therapy developing. This novel treatment has great potential， butmany issues need to be solved before cancer patients can be safely and efficiently treated. Thoseissues like specific， efficient transfection/transduction， toxicity and so on. Gene therapymeans transfer the extenal gene to cells，corret the heredity and get impairment of gene.After20-year research， there was great progression to treat the heredity disease，metabolism diseaseand malignant tumor using gene therapy，and some treatments have been used in clinical case.In the current study， we tried to use our novel developed nanoparticale system to addressspecific， efficient transfection/transduction and toxicity issues during cancer gene therapy.
TRAIL belongs to the tumor necrosis factor (TNF)-cytokine superfamily. TRAIL inducesapoptosis in a wide variety of transformed cell lines. The killing activity of TRAIL iscancer-specific and has little or no effect on most normal cells and tissues. Although themechanism for this cancer-specific action of TRAIL is poorly understood， it is recognized thatTRAIL has the potential to become a promising antitumor therapy reagent and may provide anovel approach for cancer treatment.The human telomerase reverse transcriptase (hTERT) ishighly active in85–90%of human cancer， while its activity is lower or undetactable in mosthuman normal somatic cells. The hTERT promoter was considered as a tumor specific promoter.Many studies have used the hTERT promoter to drive antitumor gene expression selectively incancer cells， without or little affecting normal cells to restrict or limit unwanted side effects.Our previous study demonstrated that adenoviral vector encoded human TRAIL， driven by the hTERT promoter， could efficiently mediate apoptosis of an ACC tumor model in vivo. Weused the plasmid pACTERT-TRAIL.
Recently medical nanotechnology has developed quickly and is already employed in manyclinical applications. These multifunctional nanoparticles become very promising for manyapplications. Nano-or micro-particles provide specific surface for many kind of molecules tobind， such as chemical compounds or DNA molecules (on the surface or in the center， bothmethods are exit). Magnetic nanoparticle are a special kind of nanoparticle， which has beenused in drug delivery and rapid biological separation. Several previous studies demonstrated thatDNA molecules could bind to magnetic nanoparticles by non-covalent bonds and used this toincrease transduction efficiency by applying a magnetic field around tumor.
To overcome treatment limitations， we developed a novel treatment combining genetherapy and nanotechnology. In this study， we created a plasmid pACTERT-TRAIL， whichused the human telomerase reverse transcriptase promoter，a tumor specific promoter，to drivea TRAIL. A Fe_3O_4-PEI-plasmid complex (FPP) was generated， in which the Fe_3O_4nanoparticles modified by positive charge polyethylenimine to be able to carry the negativecharge plasmid.Ｗe used polymer modified Fe_3O_4nanoparticles as vector，which is prepared byalkaline coprecipitation in the presence of the cationic polymer PEI. The amine groups on thesurface of nanoparticles can combine with the phosphate radical of plasmid DNA，pACTERT-TRAIL， by electrostatic interaction to form nanocomposite particles. This non-viraldelivery system could be more safe with less side effects. The magnetism of nanocompositeparticles could locally target the DNA delivery. The PEI polymer can protect the DNA fromdigesting in endosome due to their buffer capacity so-called―proton sponge mechanism‖. FPPinteract with the negative charges of the cell membrane facilitating entry into the cells. A studysuggested that positive molecules interact with cells strongly leading to faster cellular uptakecompared to neural and negative molecules.
pACTERT-TRAIL transfected with the PEI modified iron oxide nanoparticles with aNd-Fe-B magnet into SACC-83cells，the proliferation and apoptosis of SACC-83weredetected by MTT，FCM techniques. In vivo， after FPP was injected into tumors of ACC innude mice，the tumor volumes，the morphology of organs and elements of blood in nude mousewere detected， in order to illuminate the function of the pACTERT-TRAIL.The grouptransfected with the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet had the highest transfection efficiency. The results from MTT assay and Annexin V-FITC assayindicated that the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet couldefficiently deliver functional plasmids into the target cells. The group of PEI modified ironoxide nanoparticles with a Nd-Fe-B magnet inhibit the tumor growth of adenoid cysticcarcinoma and there were no obviously change of morphology of organs and elements of bloodin the nude mice.
Human TRAIL has a selective tumor-specific killing activity and little or no effect on mostnormal cells and tissues.Previous studies， however， showed that high concentration ofTRAIL could induce toxicity of hepatocytes and human brain cells in vitro. The TRAIL toxicitieshave potential ischemic and hemorrhagic reaction in normal tissues. To minimize the TRAILpotential side effects and restrict the expression of TRAIL in the cancer cells， we selected thehTERT promoter as a cancer specific promoter. Our previous study had shown that the hTERTpromoter selectively mediated the expression of TRAIL in SACC-83tumor cells resulting in thespecific killing of these cells in vitro. In present study， we used the PEI modified iron oxidenanoparticles to deliver therapeutic plasmid into target cells and employed a Nd-Fe-B magnetaround the target at the same time to further restrict the transfection occurred in the target side.Therefore， the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet provides asimple， specific， efficient and less toxic transfection method in vitro and in vivo compared tothe adenoviral vector.
In summary， this study demonstrated that we were able to create a useful PEI modifiediron oxide nanoparticles， which could link plasmid DNA and efficiently transfected thetherapeutic plasmid into the ACC tumor cells， SACC-83， in vitro and in vivo in a specificmanner using a Nd-Fe-B magnet around the target site. This PEI modified iron oxidenanoparticles show the possible clinical potential to treat the ACC in near future.
肿瘤坏死因子相关凋亡诱导配体TRAIL属于肿瘤坏死因子超家族。TRAIL可以诱导很多肿瘤细胞系凋亡。TRAIL的杀伤作用是肿瘤特异性的，对正常细胞及组织几乎没有副作用。虽然对于这种肿瘤特异性杀伤性作用的机制几乎不知道，但是TRAIL仍被认为是肿瘤治疗可能的有效的治疗肿瘤的有效基因，能为肿瘤治疗提供新的思路。人端粒酶逆转录酶(hTERT)在85-90%的恶性肿瘤中高表达，而在正常体细胞中几乎没有表达。所以hTERT被认为是肿瘤转基因治疗的特异性启动子。许多研究都将hTERT启动子使抗肿瘤基因在肿瘤细胞中靶向表达，对正常细胞没有杀伤作用，减少了其副作用。我们之前的研究表明，腺病毒载体编码TRAIL基因，并由hTERT启动子介导在体内可以有效杀伤ACC。本实验中我们设计使用质粒载体pACTERT-TRAIL。
近年来，纳米技术飞速发展，并在很多医学领域有广泛的应用，尤其是多功能的纳米微粒的医学应用。纳米或者微米颗粒的表面可以与许多分子结合，例如DNA分子。磁性纳米微粒是一类特殊的纳米微粒，已经被用于药物输送、快速生物分离等方面。一些以前的研究表明DNA分子能通过共价键与磁性纳米微粒复合，在外加磁场作用下可以提高转染效率。
在国家自然科学基金的资助下（30672338、30740420551、30830108），我们将转基因治疗与纳米技术联合开发了一种新的治疗方法。合成了以人端粒酶逆转录酶为启动子，表达肿瘤坏死因子相关凋亡诱导配体TRAIL的质粒载体pACTERT-TRAIL。此外合成了带正电的聚合物PEI修饰Fe_3O_4纳米微粒，使其与带负电的质粒载体复合，形成了Fe_3O_4-PEI-质粒复合物(FPP)。我们在碱性共沉淀方法里面加入了分枝状聚合物PEI，合成了PEI修饰Fe_3O_4纳米微粒作为载体。纳米微粒表面的胺基可以与质粒DNA的磷酸根通过静电作用复合形成含DNA的FPP。非病毒载体系统更加安全具有更小的副作用。它可以局部靶向输送DNA。溶酶体内聚合物PEI通过质子海绵效应保护DNA不被降解。并且FPP带正电可以与带负电的细胞膜复合，从而更快的进入细胞。研究表明：与带负电和中性的粒子相比，带正电的粒子可以与细胞结合从而更快的被细胞吞噬。
PEI修饰的Fe_3O_4磁性纳米微粒组在Nd-Fe-B外加磁场作用下，将pACTERT-TRAIL转染SACC-83细胞，通过MTT、流式细胞仪等技术，观察其对SACC-83增殖和凋亡作用；体内以腺样囊性癌裸鼠移植瘤模型为研究对象，外加磁场作用下磁转染pACTERT-TRAIL，测量肿瘤的大小、检测目的基因的表达以及观察裸鼠脏器形态学和血液学等改变，探讨pACTERT-TRAIL对实验动物体内腺样囊性癌的治疗作用。结果发现，体外转染实验中，PEI修饰的Fe_3O_4磁性纳米微粒组在Nd-Fe-B外加磁场作用下，获得了更高的转染效率。MTT实验及Annexin V-FITC/PI双染测定SACC-83细胞的凋亡比率结果表明，PEI修饰的Fe_3O_4磁性纳米微粒组在Nd-Fe-B外加磁场作用下更加有效的杀伤靶细胞。体内实验中，磁性纳米载体组在外加磁场作用下，能显著抑制腺样囊性癌裸鼠移植瘤生长，并且荷瘤裸鼠的心脏、肝脏、肾脏、肺脏，未见明显的形态学改变，全血中红细胞、白细胞、血小板数目，血红蛋白含量也未见明显变化。
人TRAIL具有选择性的肿瘤特异性杀伤活性，而对大多数正常细胞和组织几乎没有杀伤作用。然而以往的研究结果表明，高浓度的TRAIL在体外可诱发肝细胞和人脑细胞的毒性。TRAIL的毒性还包括对正常组织具有潜在的缺血性和出血性反应。为了尽量减少对TRAIL潜在的副作用，并限制在正常细胞中的表达，我们选择了hTERT启动子作为恶性肿瘤特异性启动子。我们以前的研究曾显示，hTERT启动子选择性介导的TRAIL在SACC-83中表达并有效杀伤了癌细胞。这些细胞的特异性杀伤肿瘤细胞作用中，我们使用的PEI修饰的氧化铁纳米粒子输送质粒进入靶细胞，通过在外加的Nd-Fe-B磁场作用下，可以将纳米质粒复合微粒进一步限制在肿瘤细胞。因此，PEI修饰的Fe_3O_4磁性纳米微粒在Nd-Fe-B磁场作用下与腺病毒载体相比更加简单，具体，高效，低毒性的转染方法。
总之，这项研究表明，我们能够创建一个有效的PEI修饰的Fe_3O_4磁性纳米微粒，它可以连接质粒DNA，在磁场作用下可以有效地转染ACC的肿瘤细胞SACC-83，体内外均取得了较好的抑制肿瘤的作用。PEI修饰的Fe_3O_4磁性纳米微粒治疗具有很大的临床应用潜力。
ACC is one of the most common malignant neoplasms in the human salivary gland，representing~10%of salivary gland tumors. Adenoid cystic carcinoma is the second mostcommon malignant tumor in the salivary gland.The ACC grows relatively slowly and has a poorprognosis compared to other malignant tumors in salivary glands. However， the currenttreatments for ACC，including surgery， radiation therapy， chemotherapy and combinationsthereof，still could not improve overall mortality rates. To efficiently and successfully treatACC， new treatments are needed. To date， cancer is still one of major lethal diseases inhuman for which treatment can be problematic. Over decade ago， cancer gene therapy startedto develop with the gene therapy developing. This novel treatment has great potential， butmany issues need to be solved before cancer patients can be safely and efficiently treated. Thoseissues like specific， efficient transfection/transduction， toxicity and so on. Gene therapymeans transfer the extenal gene to cells，corret the heredity and get impairment of gene.After20-year research， there was great progression to treat the heredity disease，metabolism diseaseand malignant tumor using gene therapy，and some treatments have been used in clinical case.In the current study， we tried to use our novel developed nanoparticale system to addressspecific， efficient transfection/transduction and toxicity issues during cancer gene therapy.
TRAIL belongs to the tumor necrosis factor (TNF)-cytokine superfamily. TRAIL inducesapoptosis in a wide variety of transformed cell lines. The killing activity of TRAIL iscancer-specific and has little or no effect on most normal cells and tissues. Although themechanism for this cancer-specific action of TRAIL is poorly understood， it is recognized thatTRAIL has the potential to become a promising antitumor therapy reagent and may provide anovel approach for cancer treatment.The human telomerase reverse transcriptase (hTERT) ishighly active in85–90%of human cancer， while its activity is lower or undetactable in mosthuman normal somatic cells. The hTERT promoter was considered as a tumor specific promoter.Many studies have used the hTERT promoter to drive antitumor gene expression selectively incancer cells， without or little affecting normal cells to restrict or limit unwanted side effects.Our previous study demonstrated that adenoviral vector encoded human TRAIL， driven by the hTERT promoter， could efficiently mediate apoptosis of an ACC tumor model in vivo. Weused the plasmid pACTERT-TRAIL.
Recently medical nanotechnology has developed quickly and is already employed in manyclinical applications. These multifunctional nanoparticles become very promising for manyapplications. Nano-or micro-particles provide specific surface for many kind of molecules tobind， such as chemical compounds or DNA molecules (on the surface or in the center， bothmethods are exit). Magnetic nanoparticle are a special kind of nanoparticle， which has beenused in drug delivery and rapid biological separation. Several previous studies demonstrated thatDNA molecules could bind to magnetic nanoparticles by non-covalent bonds and used this toincrease transduction efficiency by applying a magnetic field around tumor.
To overcome treatment limitations， we developed a novel treatment combining genetherapy and nanotechnology. In this study， we created a plasmid pACTERT-TRAIL， whichused the human telomerase reverse transcriptase promoter，a tumor specific promoter，to drivea TRAIL. A Fe_3O_4-PEI-plasmid complex (FPP) was generated， in which the Fe_3O_4nanoparticles modified by positive charge polyethylenimine to be able to carry the negativecharge plasmid.Ｗe used polymer modified Fe_3O_4nanoparticles as vector，which is prepared byalkaline coprecipitation in the presence of the cationic polymer PEI. The amine groups on thesurface of nanoparticles can combine with the phosphate radical of plasmid DNA，pACTERT-TRAIL， by electrostatic interaction to form nanocomposite particles. This non-viraldelivery system could be more safe with less side effects. The magnetism of nanocompositeparticles could locally target the DNA delivery. The PEI polymer can protect the DNA fromdigesting in endosome due to their buffer capacity so-called―proton sponge mechanism‖. FPPinteract with the negative charges of the cell membrane facilitating entry into the cells. A studysuggested that positive molecules interact with cells strongly leading to faster cellular uptakecompared to neural and negative molecules.
pACTERT-TRAIL transfected with the PEI modified iron oxide nanoparticles with aNd-Fe-B magnet into SACC-83cells，the proliferation and apoptosis of SACC-83weredetected by MTT，FCM techniques. In vivo， after FPP was injected into tumors of ACC innude mice，the tumor volumes，the morphology of organs and elements of blood in nude mousewere detected， in order to illuminate the function of the pACTERT-TRAIL.The grouptransfected with the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet had the highest transfection efficiency. The results from MTT assay and Annexin V-FITC assayindicated that the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet couldefficiently deliver functional plasmids into the target cells. The group of PEI modified ironoxide nanoparticles with a Nd-Fe-B magnet inhibit the tumor growth of adenoid cysticcarcinoma and there were no obviously change of morphology of organs and elements of bloodin the nude mice.
Human TRAIL has a selective tumor-specific killing activity and little or no effect on mostnormal cells and tissues.Previous studies， however， showed that high concentration ofTRAIL could induce toxicity of hepatocytes and human brain cells in vitro. The TRAIL toxicitieshave potential ischemic and hemorrhagic reaction in normal tissues. To minimize the TRAILpotential side effects and restrict the expression of TRAIL in the cancer cells， we selected thehTERT promoter as a cancer specific promoter. Our previous study had shown that the hTERTpromoter selectively mediated the expression of TRAIL in SACC-83tumor cells resulting in thespecific killing of these cells in vitro. In present study， we used the PEI modified iron oxidenanoparticles to deliver therapeutic plasmid into target cells and employed a Nd-Fe-B magnetaround the target at the same time to further restrict the transfection occurred in the target side.Therefore， the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet provides asimple， specific， efficient and less toxic transfection method in vitro and in vivo compared tothe adenoviral vector.
In summary， this study demonstrated that we were able to create a useful PEI modifiediron oxide nanoparticles， which could link plasmid DNA and efficiently transfected thetherapeutic plasmid into the ACC tumor cells， SACC-83， in vitro and in vivo in a specificmanner using a Nd-Fe-B magnet around the target site. This PEI modified iron oxidenanoparticles show the possible clinical potential to treat the ACC in near future.
引文
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