Bionic Fabrication Of Dynamic Biointerfaces And Study On The Modulation Of Cell Adhesion

Dynamic receptor–ligand interactions between cells and the extracellular matrix(ECM)are crucial to physiological and pathological cellular behaviors in vivo.The construction of dynamic biological surface material(i.e.,dynamic biointerface)to realize controllable cell behavior regulation have many potential applications in fundamental biology,regenerative medicine,disease treatment and other fields by modulating the receptor-ligand interactions in ECM.As a kind of extracellular matrix material,dynamic biointerface can simulate the interaction between cells and extracellular matrix material in vitro through stimulus response methods,which has important theoretical and practical significance for the development of biological materials.Therefore,develop a low-toxic or nontoxic biological stimulus-responsive multifunctional dynamic biointerface for dynamically regulating cell adhesion has a significance role in basic biology and cell based clinical diagnosis.This paper focused on the construct of biological interface with dynamic bioactivity through intrinsic biological stimulation(such as blood glucose fluctuation).Based on the reversible catechol-boronate dynamic covalent chemistry,a series of dynamic bioactive interfaces with sugar response performance were obtained by designing different types of mussel inspired peptides.Various characterization techniques were used to analyze the morphology,chemical composition,biotoxicity and reversible molecular release of the dynamic biointerfaces,at last studied the application to the regulation of“adhesion and desorption”,“capture and release”processes for different cells and bacteria,and tested the performance in cell adhesion modulation and tumor cell capture and isolation.The main research works were shown as follows:(1)Constructed a macro dynamic bioactive surface for reversible cell attachment and selective cell recognition based on the phenylboronic acid-catechol dynamic covalent interaction.Prepared a phenylboronic acid(PBA)-containing polymer PHEAA-co-PAFPBA grafted substrate by photo-initiated polymerization on quartz slide,and designed bioactive mussel inspired peptides((DOPA)₄-Y-X)with a catechol-containing sequence and a cell-binding sequence at each end.The bioactive mussel-inspired peptides were then reversibly bound on the PBA-containing polymer-grafted substrate through sugar-responsive catechol-boronate covalent interactions.Specifically:a dynamic biointerface containing RGD sequence by mussel inspired peptide(DOPA)₄-S₅-GRGDS was used to dynamic modulate the stem cell BM-MSC adhesion behaviors,and a dynamic biointerface containing tumor targeting peptide Wx EAAYQr FL sequence by mussel inspired peptide(DOPA)₄-S₅-Wx EAAYQr FL was used to capture and release of tumor cell MCF-7.The results showed that:the dynamic biointerfaces have a excellent selective performance for both adhesion and capture efficiencies to the targeted cells;and the PBA polymer grafted substrate without bioactive molecules basically did not adhesion or capture cells,showing a good anti-unspecific adsorption ability;the cells adhered or captured by the dynamic interface could be released by the treatment of 60 m M fructose solution,and released more than 90%adhered cells after 20 minutes,showing excellent sugar release performance.(2)Constructed a magnetic micro dynamic biointerface for magnetic separation of tumor cells based on the phenylboronic acid-catechol dynamic covalent interaction.fabricated a dynamic magnetic micro platform on micro magnetic beads(MMBs),demonstrated a paradigm shift of dynamic biointerfaces from macroscopical substrates to micron-sized particles by reversible engineering a PBA-functionalized magnetic microbead with mussel-inspired cancer-cell-targeting peptide(DOPA)₄-S₅-Wx EAAYQr FL,for selective isolation of tumor cell MCF-7.Our results demonstrated that the dynamic magnetic platform was capable for isolate of MCF-7 cells both from cell culture medium and real blood samples.the magnetic micro platform could capture more than 78%from 10⁴cells/ml of MCF-7 cells by incubation for 60 min,and capture more than 90%cells when reduced the cell density from 10⁴cells/ml to 20 cells/ml;while HL60 and Hep G 2 cells could hardly be captured by using this platform;more than 93%of captured cells can be released after fructose treatment for 30 min;the released cells shown high biological activities and good proliferation after 5 days of continuous culture;at last,about 20%of tumor cells can be separated from the artificial blood samples,which has a good tumor recognition ability in real blood samples.(3)Constructed a multifunctional dynamic biointerface based on reversible covalent and biorthogonalization for the universality separation of biological particles.Reported a dynamic biomaterial interface with optional and diversified bioactivities by the combination of reversible catechol-boronate and bioorthogonal click chemistry.Concretely,clickable mussel-inspired peptides(DOPA)₄-S₄C-(DBCO)and N₃-capped bioactive moieties(small molecular saccharide mannose,macromolecular peptides Wx EAAYQr FL and DNA aptamers Sgc8)were designed for on-demand and reversible binding on biomaterial interfaces,enabled reversible presentation of diversified bioactivities,exhibiting the multipotential to manipulate a variety of cell-biomaterial interactions.this system could be efficiently used for reversible and selective binding of different cancer cells such as MCF-7,CCRF CEM and even bacterial microorganism E.coli.The results shown that:the dynamic bioactive interfaces shown excellent selective capture,separation and release abilities for E.coli,MCF-7 and CCRF CEM cells;and can be reused after regeneration;has good biological activities after capture and release cycle for both cells and bacteria;at last,the dynamic biological interface platform can capture more than 12%of cancer cells from artificial blood,showing the effective capture efficiency in real blood samples.Overall,our work provides not only a facile strategy for mimicking the multipotential of natural ECM,but also a versatile dynamic biointerface with promising prospects in biomedical science,in particular,the cell isolation for diagnostics and therapeutics.(4)Constructed an electrochemical cell sensor based on dynamic biointerface meaterila for the specific detection of tumor cells.fabricated an electrochemical dynamic biosensor on ITO conductive glass for selective recognition,separation,detection and release of tumor cells.The captured tumor cells were quantitatively analyzed by electrochemical methods.The experimental results shown that a linear relationship could be established between the captured cell densities and impedances(or currents)by EIS(or DPV)methods,with the cell capture density per unit area in the range of 10 cells/mm² to 66 cells/mm²,the minimum detection limit was 3 cells/mm².The results of electrochemical methods(EIS/DPV)in the mixture solution of MCF-7 and Hep G 2cells shown no statistical difference to those of fluorescent labeling method.the capture and release of cells on this sugar responsive biosensor was noninvasive with the cell viability over 87.2%.The excellent performances of electrochemical dynamic biosensor reported in this paper would be a novel and promising tool for separation,detection and release of biomarkers.