发展基于细胞相容的三维纳米界面的循环肿瘤细胞的超低损失捕获和释放及高纯度分离技术

The capture and isolation of rare circulating tumor cells (CTCs) has attracted a great deal of research interest in recent years. However, there are still some challenges including purity as well as viability of the captured CTCs. Here，we will develop high effective platforms for the isolation of CTCs of high purity without losing cell number and viability using cellular compatible nanomaterials, proper molecular design on the interface, and non-destructive release strategy of the captured CTCs. Chitosan 3D nanostructured surface is firstly fabricated by electrospraying or electrospinning to provide a cellular compatible interface for specific capture of viable CTCs. The morphology of the deposited particles or fibers was varied depending on the conditions of electrospraying or electrospinning and chitosan solution. To obtain high capture specificity, antifouling molecules as well as DNA aptamer are introduced onto the interface simultaneously. On one hand, the antifouling molecules, such as PEG, CBMA polymer, PNIPAM, will effectively improve hydrophilic of the interface and decrease the nonspecific adhesion of blood cells. CBMA and NIPAAm will be grafted onto the surface of the chitosan nanoparticles or nanofibers by atom transfer radical polymerization (ATRP). On the other hand, a DNA aptamer against EpCAM is employed to be coupled with the antifouling molecules to target and capture CTCs specifically. The further purification of the captured rare number CTCs is realized through in-situ culture of the captured cells on the surface of the chitosan 3D nanostructures. Furthermore, release of aptamer-captured cells can be accomplished by methods such as complementary DNA strand and temperature change. CBMA polymer is of excellent hydrophibicity which makes it feasible to control the nonspecific interaction between cells and chitosan nanofibers. PNIPAM is a well-known thermo-responsive polymer that shows an extended hydrophilic chain conformation below its LCST (~32 °C) in aqueous solution and undergoes a phase transition to insoluble and hydrophobic aggregates above the LCST. By varying the surrounding temperature, the platform can capture cells from blood or the mixture of cells at 37℃, and then the release nonspecific cells at 4℃. The targeted cancer cells are then released by complementary strand at 4℃. The development of our general bio-platform is expected to obtain CTCs with high purity and keeping alive for the subsequent molecular characterization and functional analysis, which is of great significance. It is expected to reveal the basic mechanism of the harmonic effect from cellular compatible nanostructures, antifouling molecules, the specific capturing by aptamer. The general conclusions will be obtained for the isolation of the viable CTCs with high purity and the non-destructive release of the captured CTCs on cellular compatible chitosan nanostructured surface.

发展创新技术以实现CTC数量与生物活性超低损失捕获和释放及CTC高纯度分离对后续CTC分子鉴定及功能分析研究具有极其重要意义。本研究尝试从“材料选择、结构构筑、界面分子设计、释放策略”四个方面设计以达到此目的。首先，选择细胞相容性好的壳聚糖构筑三维纳米结构基底，在纳米界面上进行分子设计，利用抗粘附分子对血细胞非特异吸附的抑制与适配体对CTC特异捕获的协同作用，实现CTC在数量和生物活性上超低损失捕获和提高CTC的捕获纯度。随后通过温度变化及互补链两种方案对适配体进行调控实现所捕获CTC在数量和生物活性上超低损失释放。本研究所发展的基于纳米结构匹配和界面分子协同作用的高效通用捕获平台，有望获得高纯度、良好生物活性的CTC样品。通过这一研究，有望揭示出细胞相容性纳米结构、抗粘附分子、适配体对CTC高特异捕获的协同效应的内在机理，归纳出壳聚糖纳米界面上CTC高纯度分离和超低损失释放的一般规律。

循环肿瘤细胞（CTC）在癌症转移中的决定作用已引发人们的广泛关注，CTC检测在癌症病人的诊断、治疗和监控等整个就医周期中都有重要的价值和意义。为了便于后续的CTC分子鉴定及功能分析研究，发展创新技术以实现CTC数量与生物活性超低损失捕获和释放及CTC高纯度分离具有极其重要的意义。本研究从“材料选择、结构构筑、界面分子设计、释放策略”四个方面设计以达到此目的。一方面，选择细胞相容性好的壳聚糖构筑三维纳米结构基底，并在纳米界面上进行分子设计，利用羧酸甜菜碱（CBMA）、N-异丙基丙烯酰胺（NIPAM）等抗粘附分子对血细胞非特异吸附的抑制与适配体对CTC特异捕获的协同作用，实现CTC在数量和生物活性上超低损失捕获和提高CTC的捕获纯度。随后通过温度变化及互补链两种方案对适配体进行调控实现所捕获CTC在数量和生物活性上超低损失释放。另一方面，基于磁分选技术，选择便于分离的磁性纳米粒子构筑纳米结构，并修饰一层CBMA或SBMA分子，利用其极好的细胞相容性和抗粘附性能，减少血细胞等对CTC捕获的干扰；并进一步修饰单抗体或双抗体捕获分子，实现了CTC的高效、高纯度捕获、计数和释放。基于纳米结构匹配和界面分子的协同作用，所发展的高效通用捕获平台和释放策略，能够获得高纯度、良好生物活性的CTC样品。目前对于模型细胞系已得到了较理想的结果，并继续深入开展了对临床病人样本的检测工作。通过这一研究，揭示出细胞相容性纳米结构、抗粘附分子、亲和分子（适配体、单/双抗体）对CTC高特异捕获的协同效应的内在机理，归纳出壳聚糖纤维、磁性纳米粒子等纳米界面上CTC高纯度分离和超低损失释放的一般规律。