【作者】 夏洁；

【导师】 刘宝瑞； 钱汉清；

【作者基本信息】 南京大学 ， 临床医学（肿瘤学）（专业学位）， 2017， 硕士

【摘要】 背景:近年来随着生物膜材料的兴起,红细胞膜、白细胞膜、血小板膜相继被用于构建纳米药物载体。由于膜蛋白能够保留于提取的细胞膜上,因此所构建的纳米载体仍然具有其来源细胞的生物特性。例如红细胞膜包裹的纳米粒子具有体内长循环特性,白细胞膜纳米粒子具有向炎症驱化的特性等。与脂质体相比,细胞膜为纳米药物载体赋予了特殊的生物特性。因此,利用肿瘤细胞膜实现抗肿瘤药物的靶向投递是目前研究的热点。与此同时,肿瘤细胞因其膜上所表达的多种细胞粘附分子而被广泛研究。这些膜上黏附分子促使肿瘤细胞粘附成团并促进转移,抵抗失巢凋亡。同源的肿瘤细胞经常在体内黏附成肿瘤团块,这种特别的黏附现象被研究者们称作“同源细胞黏附”或者“自我黏附”。多数研究也已在体外证实了肿瘤细胞的这种自我黏附特性。之后,研究者们进一步研究探讨发现,正是肿瘤细胞膜上的黏附蛋白尤其是E-钙黏素介导了同源肿瘤细胞之间的紧密链接。因此,黏附蛋白是形成同源细胞黏附的主要原因。得益于目前成熟的生物膜载体制备工艺,保留有黏附蛋白的肿瘤细胞膜被应用于构建仿生纳米药物载体。通过大量实验研究发现,这种新型生物膜纳米载药系统具有自我选择的靶向性,即同种肿瘤细胞膜包裹的纳米粒子可以优先靶向到同种肿瘤细胞上。这种特性被称作“同源靶向性”。然而,由于黏附蛋白表达的普遍性,例如CEA、NCAM、E-cadherin等常常表达在多种肿瘤细胞或正常细胞表面。因此,表达有相同黏附分子的异源肿瘤细胞或与正常细胞间便会产生错误识别。并且,有团队研究肿瘤细胞外泌体时发现,同源肿瘤细胞外泌体并未在细胞及小鼠实验中表现出明显的同源靶向性。因此综上所述,仍需进一步研究肿瘤细胞膜的同源靶向性及其影响因素,以进一步优化基于肿瘤细胞膜纳米药物投递系统的靶向性,提高抗肿瘤药物的疗效。目的:构建肿瘤细胞膜纳米载体,评价其肿瘤靶向性。方法:(1)本实验设计出一种新型肿瘤细胞膜纳米载体系统。该系统以PLGA纳米粒子为内核,核内负载香豆素-6荧光探针,外部包覆一层肿瘤细胞膜。(2)通过将该纳米粒子分别与同源、非同源肿瘤细胞(包括MKN45细胞、AGS细胞、Hela细胞)、无限增殖细胞(包括HBE细胞、HELF细胞)共孵育,利用流式细胞分析仪、激光共聚焦显微镜、荧光酶标仪研究细胞对纳米粒子的摄取,评价同源之间和异源之间纳米粒子的靶向性。(3)通过体外3D细胞培养验证不同肿瘤细胞的黏附能力,Western blot及免疫荧光成像分析上述肿瘤细胞膜上E-钙粘素、N-钙粘素、CEA的表达情况,并研究肿瘤细胞膜纳米粒子靶向性的影响因素及相关机制。结果:本文所涉及的细胞膜纳米粒(CCNP)均未表现出优先的自我靶向能力。在几乎所有细胞系中,MKN45细胞膜纳米粒(MKN45-NPs)相较AGS(AGS-NPs)及Hela纳米粒(Hela-NPs)表现出更强的细胞靶向能力。通过3D细胞培养实验发现,MKN45细胞具有最强的自我粘附能力。进一步进行蛋白分析发现,MKN45细胞及HBE细胞大量表达E-钙粘素。Hela细胞及HBE细胞表达大量N-钙粘素。CEA则普遍表达在本实验所有细胞系中。结论:并非所有的肿瘤细胞膜载体均具有同源靶向性。但E-钙黏素是影响肿瘤细胞膜纳米粒子靶向性的重要因素。由E-钙黏素高表达的肿瘤细胞膜构建的纳米粒子不但可以优先靶向到E-钙粘素高表达的肿瘤细胞上,还可以靶向到粘附性低的肿瘤细胞上更多还原

【Abstract】 Background:In recent years,with the rapid development of biological membrane materials,not only erythrocyte membrane,white blood cell membrane but also platelet membrane have been studied and applied in the field of drug delivery.After a complex membrane extraction step,the protein on the cell membrane remains intact.After repeated studies,it was confirmed that those membrane vehicles had the biological characteristics as their source cells.For example,the red cell membrane coated nanoparticles have accomplished the long circulation in vivo just as erythrocyte;and the white cell membrane nanoparticles still have the characteristics of driving to inflammation.Compared with the conventional liposome,those bio-membrane package has given the nano-drug delivery field with special biological activity.However,although the cell membrane carrier has achieved the corresponding biological function,but it has not shown more in the field of tumor targeted drug delivery.At the same time,the tumor cells are considered to have the characteristics of homologous adhesion due to the various adhesion molecules expressed on the membrane.It is homologous adhesion that can help tumor cells accumulate into cell aggregates,promote metastasis and resist anoikis.Most of the studies have confirmed such adhesion propertyof tumor cells in vitro.Besides,it is also found that the cancer cell membrane adhesion proteins especially E-cadherin,mediate the adhesion between adjacent cancer cells.Therefore,some researches have stripped cancer cell membrane with adhesion proteins reserved and applied as nano-drug vehicles.To their gratification,such new nano-drug delivery system has achieved selective self-target and obtained effective therapy results in vivoHowever,the adhesion proteins on tumor cell membrane are often expressed on many kinds of cancer cells,such as CEA,NCAM,E-cadherin and so on.The adhesion between different cancerous cells may takes plays.The cancer cell membrane vehicles may loss its selective homotypic targeting capacity.Moreover,it has been found that no obvious homotypic targeting property is shown on exsomes derived from homologous cancer cells.Hence,it is necessary to make studies on the targeting property of cancer cell membrane vehicles.Objective:In order to study exact homotypic targeting capacity of cancer cell membrane vehicles,and investigate the possible mechanism.Methods:In this study,we firstly designed a novel drug delivery system synthesized with a cancer cell membrane carrier and a coumarin-6 fluorescent probe loaded PLGA nanosphere core(CCNPs).Here,the membrane of MKN45 cells,AGS cells and Hela cells were used to form its corresponding CCNPs.Secondly,though strict quality control,such CCNPs were co-cultured respectively with homologous and non homologous tumor cells(including MKN45 cells,AGS cells and Hela cells),immortalized cell(including Hbe cells,Helf cells).The results were obtained by flow cytometry,confocal microscopy and fluorescence microplate in vitro.Furthermore,the self-adhesion capacity of different cancer cells was verified by 3D cell culture in vitro.Finally,Western blot analysis and immunohistochemical analysis of several adhesion proteins were used to analyze the effect of targeting.Results:Our results revealed that MKN45 cell membrane coated nanoparticles(MKN45-NPs)were more adhesive to almost all cells than AGS-or Hela-NPs,suggesting that adhesive abilities of each cancer membrane were different and heterogenous CCNPs rather than homologous CCNPs might preferentially target to some cancer cell.Moreover,a non-specific adhesion ability of those CCNPs had been found when membrane cloaking did not enable MKN45-NPs and other CCNPs to selectively target to their source cells.Then further investigations implied that adhesion abilities of CCNPs were inherited from their mother cells,and significantly affected by E-cadherin expression.And CCNPs’ uptake was also related to the surface CAMs of the recipient cells.Conclusion:Not all the cancer cell membrane coat holds homotypic targeting propety E-cadherin highly expressed cancer cell membrane carrier shows a stronger targeting,which can help target to the low-adhesive tumor cells.And,E-cadherin-present CCNP can preferentially targets to E-cadherin high-expressed tumor cells.Hence,it’s also an opportunity that membranes of some specific tumor cells can be used to deliver cargos to many other tumor cells.更多还原