Fabrication And Properties Of Nanocage Featured Material For Circulating Tumor Cells Capture

Circulating tumor cells(CTCs)are tumor cells that shed from the primary tumor and then circulate through the peripheral blood circulation.Efficient CTCs-capture technologies have important implications for early diagnosis,prognosis monitoring,pharmacodynamics evaluation and personalized treatment of cancer.Due to the rarity and heterogeneity of CTCs,the specific biomolecules-depended CTCs isolation methods suffer from the disadvantages of poor broad-spectrum and false negatives.To develop efficient and broad-spectrum CTCs capture materials,we propose a new strategy of label-free capture of CTCs based on the size and structure matching effect between novel "nanocage" and CTCs filopodia.Inspired by the structural diversity of natural pollen,we attempted to fabricate the CTCs filopodia matched micro-nano structures from natural pollen exine.We obtained micro-nano structured particles by etching and chemical modification with pollen exines,which were further prepared into micro-nano structured films for CTCs capture.By investigating the effects of different types and sizes of micro-nano structures on CTCs capture,we proposed the mechanism of efficient CTCs capture with nanocage and established a theoretical and technical foundation for developing nanomaterials for label-free CTCs capture.The research concrete content and results are as follows:(1)To overcome the drawbacks of hydrophobicity,autofluorescence and the covered surface pattern of natural pollen,we took Chrysanthemum(Chry)pollen as a model to study the etching and modification effect of concentrated H2SO4 on pollen exine.The results showed that the natural micro-nano structures of pollen exine were fully exposed after etching with concentrated H2SO4.Meanwhile,the increase of-SO3H group and the decrease of C=O Group on the surface of particles lead to the increasing hydrophilicity and autofluorescence quenching.The treatment time of concentrated H2SO4 was further optimized.The results showed that microparticles with hydrophilic surface,no autofluorescence and complete nanostructures could be obtained after 24 h of concentrated H2SO4,which could be further used for fabricating CTCs capture films.(2)The uniform and stable micro-nano structured films for CTCs capture were prepared by spraying the H2SO4 etched micro-nano particles with anhydrous ethanol and polybutyraldehyde as dispersant and binder.To select the optimal micro-nano structure for CTCs capture,Chinensis,Rose,Lotus and Chrysanthemum pollen were used to fabricate different micro-nano structured films with H2SO4 etching and spray-coating method.It was found that the hierarchical structure of Chrysanthemum pollen showed the best capture performance on MCF-7 model cancer cell line.Furthermore,we investigated the effects of Chry pollen’s different level structures on CTCs capture by examining the filopodia number,cytoskeleton distribution and focal adhesion expression.The results demonstrated that the"nanocage" structure had a decisive role in promoting CTCs capture efficiency.After 60 min of incubation,the cell-capture efficiency of nanocage film reached 91%,which surpasses the previously reported label-free nanostructured substrates.(3)To investigate the mechanism of efficient CTCs capture with nanocage,we explored the process of nanocage trapped MCF-7 cell filopodia and examined the effects of nanocage entrance diameter on CTCs capture.The results showed that the nanocage with～175 nm surface entrance.～117 nm braced frame and～166 nm interconnected cavity,matched with the cancer cell filopodia(～131 nm).Thus the filopodia anchored into the nanocage cavity and wrapped around the internal frame with an effective contact area of 0.1-0.2 μm2(enhanced 10 times than that of the reported nanostructures),leading to ultra-strong adhesion force(>22.9 nN)between the filopodia and the material and resulting in efficient CTCs capture performance.In addition,the nanocage-featured film effectively captured a broad spectrum of cancer types(MCF-7,A431,Hela,HepG2 and A549,yield～92%),proving superior and broad-spectrum CTCs capture performance.(4)To realize the application of "nanocage" featured film in clinical detection,the capture performance of CTCs in simulated blood environment was investigated.The nanocage film exhibited anti-PBMCs adhesion performance and selectively captured rare cancer cells(204000)from imitated blood samples with 106 peripheral blood mononuclear cells(～92%).Meanwhile,the nanocage film displayed good cytocompatibility,thus enabling the high survival rate(～97%)and good proliferation ability of captured cells.Furthermore,the nanocage-featured film successfully detects CTCs in blood samples taken from early-stage(4 mL)and advanced cancer patients(2 mL)suffering from various types of cancer with a detection rate of 90%(early-stage)and 100%(advanced-stage),proving the high detecting sensitivity of our novel CTCs-capture method for potential clinical practices.In summary,based on the micro-nano structure of natural pollen,we prepared highefficiency CTCs capture materials with "nanocage" structure and confirmed that the CTCs filopodia-nanocage matching effect is the main reason for efficient capture.Furthermore,the"nanocage" films were applied in clinical CTCs isolation,proving the CTCs capture sensitivity and broad-spectrum,which provides a new theoretical and technical basis for the development of CTCs capture materials for early diagnosis and cancer treatment process monitoring.