Engneering Of Cell Membrane Surface With DNA Functional Nanomaterials

Engineering of cell membrane is the use of natural or synthetic functional molecules to modify the cell membrane,so as to confer specific structure and function to cell membrane and further modulate the cell behaviors.As a new functional material,DNA nanostructures have the advantages of programmable design and high biocompatibility.DNA nanostructures provide powerful tools for the engineering of cell membrane.However,engineering of cell membrane using DNA nanostructures is still in its infancy,facing many challenges and problems.For example,the design of DNA nanostructure is complicated.Meanwhile,the interaction of DNA structures with cell membranes is not fully understood.Moreover,DNA nanostructure-mediated modulation of cell behavior is still to be developed.Therefore,in this thesis,several DNA nanostructures have been constructed for the design of artificial membrane receptor and transmembrane carriers,and their application in biochemical detection and disease treatment have been exploited.Meanwhile,the related work of liposome and DNA structure in two-phase system was also explored.The main research contents are shown as follows:1.DNA supersandwich assemblies as artificial receptors to mediate intracellular delivery of catalase for efficient ROS scavengingCatalase（CAT）is an enzyme for the degradation of H₂O₂,and can be used for the scavenging of intracellular reactive oxygen species（ROS）.However,the cellular internalization of catalase is greatly limited by the lack of specific catalase receptor expression on the cell membrane.To this end,DNA supersandwich assemblies tethered with transferrin receptor aptamer and catalase aptamer were designed,which has multivalent binding ability with catalase.This assemblies could anchor on the cell surface and serve as the membrane artificial receptor,which promotes the intracellular internalized transport of catalase and improves the intracellular ROS scavenging ability.Fluorescence imaging and flow cytometry results showed that the artificial receptor promoted cellular internalization of the enzyme with an 8-fold increase in transfection efficiency.Free radical scavenging experiments showed that catalase internalization mediated by DNA nanostructure could effectively remove intracellular ROS,reduce H₂O₂-mediated cell damage,and significantly improve cell viability.This work provides an efficient way for the cell internalization of specific protein and a new method to protect cells against ROS damage.2.Self-assembled DNA nanowires as quantitative dual-drug nanocarriers for antitumor chemophotodynamic combination therapyDNA nanostructures are biocompatible and biodegradable and have potential applications in drug delivery and disease diagnosis.The combination of chemical and photodynamic therapy is expected to improve the killing efficiency of tumor cells.Based on this,a dual drug carrier based on DNA sandwich nanowires loaded with chemotherapeutic drugs and photosensitizers was developed,and it could achieve a highly efficient cell killing by a combination of chemotherapy and photodynamic therapies.DNA supersandwich nanowires are constructed by a supersandwich hybridization of two short DNA strands.The photosensitizer Clorin E6（Ce6）is covalently connected with a short DNA chain.The chemotherapy drug DOX is inserted into the supersandwich nanowires through a non-covalent interaction.Therefore,nanowires as effective carriers of photosensitizers and chemotherapy drugs promote the internalization of drugs.DNA nanowires can be efficiently internalized into cell and the released DOX and Ce6 are eventually distributed in the nucleus and cytoplasm,respectively.Cells treated with DOX and Nanowire-Ce6 reduced cell viability to 74.5%and 55.1%,respectively.While the combined treatment of DNA nanowire-Ce6 and DOX increased cytotoxicity and decreased cell viability by 41.6%.The calculated synergistic coefficient（CI<1）indicated that PDT had a strong synergistic effect with chemotherapy.This work shows that the DNA nanowires can serve as dual-drug carrier for efficient synergistic therapeutic.It can effectively improve the killing efficiency of tumor cells and has potential application in efficient treatment of tumor.3.Design of lipophilic split aptamers as artificial carriers for transmembrane transport of adenosine triphosphateTransmembrane carrier proteins can promote mass transport and play an important physiological role in life activities.The development of artificial transmembrane carriers is expected for the modulation of transmembrane transport,and then regulation of cell physiological processes.In this work,aptamers were hydrophobic modified and anchored on the cell membrane as transmembrane artificial carriers to promote the transmembrane transport of target molecules.Adenosine triphosphate（ATP）aptamer was selected as target molecule for the design of the transmembrane artificial carrier.The aptamer was splited into two parts which modified with cholesterol at the 3’end of each sequence.Two cholesterol-modified sequences can form into artificial carriers mediated by ATP.The transport of TNP-ATP（2’（or 3’）-O-（2,4,6-trinitrophenyl）adenosine 5’-triphosphate）by artificial carriers in giant unilamellar vesicles（GUVs）showed good specificity and the transport rate was 80 n M/s.However,other water-soluble small molecule dye,such as rhodamine sulfonate and calcein,do not have permeability.At the same time,when the artificial carrier is fixed to the ATP-deficient cell membrane,it can effectively supplement the intracellular ATP,thus restoring cell vitality.In this work,hydrophobic modification of aptamer was developed for the construction of transmembrane artificial carriers,which has a good application prospect in selective transmembrane transport and drug delivery.4.Liposome-boosted peroxidase-mimicking nanozymes breaking the p H limit for H₂O₂and glucose detectionNanozyme has some unique advantages,such as resistance to harsh environment,protease degradation resistance and low cost;It shows great potential in biomedical,analytical and environmental applications.Liposomes have been used as models to study the properties of biofilms,but little is known about the effect of lipid molecules on the catalytic activity of nanozyme.Exploring of influences of liposomes on the catalytic activity of nanozyme will deepen the understanding of lipid microenvironment on the catalysis of nanozymes.In this work,Fe₃O₄and Ce O₂nanoparticles were adsorbed onto liposomes to study the effects of different charge and different composition of liposomes on the catalytic activity of nanozyme.In the presence of negatively charged liposomes,Fe₃O₄and Ce O₂showed peroxidase activity in neutral and alkaline conditions,but not in neutral and positively charged liposomes.By investigating the mechanism,it was found that negatively charged liposomes could stabilize positively charged catalytic chromogenic product ox TMB.Taking advantage of this property,we use nanozyme and liposome to the detect H₂O₂and glucose at neutral p H with detection limits of 4.6μM and 38.5μM,respectively.This finding highlights the importance of liposomes in the regulation of nanozyme activity and a way to enhance the activity of the nanozyme in neutral p H.5.Enhancing the sensitivity of DNA and aptamer probes in aqueous two-phase systemAqueous two-phase system（ATPS）is a two-phase separation system formed by polymer-polymer or polymer-salt mixture at a certain concentration and temperature.Enrichment of analytical probes or targets can accelerate the reaction and improve the sensitivity of analytical detection.In this work,a Dextran/PEG aqueous two-phase system was developed to enrich DNA probes and aptamer probes and improve the detect sensitivity of DNA,Hg²⁺and ATP.Firstly,the formation conditions of the Dextran/PEG aqueous two-phase system were investigated,and the enrichment and distribution of probes in the two phases were optimized by adjusting the salt concentration,p H value and volume ratio of the two polymers.The DNA probes and aptamer probes were mainly distributed in the Dextran phase.Under the optimal conditions,the analyte was added into the aqueous two-phase system containing the probe to detect the target.The probe maintains its secondary structure in dextran phase and retains the ability to bind to the target.Because the probe is effectively enriched,the local concentration of the probe is increased,the reaction speed is accelerated,and the fluorescence response sensitivity is increased.Compared with ordinary homogeneous solution,the detection limit is reduced by about 10 times.In this work,a simple and effective method was developed to improve the detection sensitivity of DNA probes,which based on the enrichment ability of the two-phase system for DNA probes.This work is of great value in the fields of early disease diagnosis and food safety.