Investigate Sialic Acid On Cancer Cell Surface/Light Sensitize MRSA To Membrane-targeting Antibiotics

For all mammalian cells,cell surface is decorated with a dense layer of glycan,containing glycoprotein,glycophospholipid and glycolipid,etc.Sialic acid（SA）is a family of nine-carbon anion monosaccharides that often located at the terminal of glycan chains.On cell surface,SA is chemically diverse and more than 50 chemical structures of SA in nature have been produced,which makes SA play a crucial role in a lot of crucial physiological and pathological processes.For example,the expression levels of SA are associated with a lot of diseases including brain diseases,cancers and cardiovascular diseases and others,especially its overexpression involved in cancers progression and metastasis.Therefore,developing the detection technique that can specifically trace the SA levels is significant for investigating the expression of SA,exploring the related physiological processes and assessing serious diseases.Surface-enhanced Raman spectroscopy（SERS）is based on Raman spectroscopy but can simultaneously obtain the intrinsic molecular information and process the high sensitivity.Besides,SERS technique is especially suitable for investigating the biological system owing to its distinctive advantages.For instance,it can be used to realize multiplex detection due to its narrow peaks,and multiple alternative excitation lasers allow SERS can effectively avoid the auto-fluorescence that produced by the biological tissues.Additionally,its minor damage and good photo-resistant stability make SERS suitable for investigating and analyzing the biological system.While,it stillremains challenges although SERS methods have been developed to study SA expression on cell surface.For example,developing the detection technique with no or minor damage to monitor the dynamic changes of sialic acid on cell surface.Additionally,it is still need to develop the SERS method for SA detection in clinical tissues since the overexpression of SA is the key harbinger for cancer lesions.Therefore,it is necessary that developing a SERS strategy with minor damage that can monitor the dynamic changes of SA under physiological condition and realize cancer tissue study.For the questions that still need to be solved for studying SA on cell surface,this thesis reported an approach that can study SA under physiological condition based on SERS reporter technique.The main contents are as follows:1)We designed a SERS nanoprobe that can specifically recognize SA on cell surface under the normal physiological condition based on the affinity interaction between SA and phenylboronic acid.The nanoprobe is designed by surface modification of silver nanoparticles（AgNPs）acted as SERS substrate with 4-mercaptophenylboronic acid（MPBA）that serves as the binding ligand and SERS reporter molecule.The results indicated that the changed spectral signals can be used to analyze the SA levels by testing different kinds of cell lines.We further utilized density functional theory（DFT）to explore the mechanism of recognition interaction between SA and MPBA.The SERS reporter method also was used to monitor the dynamic expression of SA with external stimuli on cancer cell lines.2)Based on the designed Ag@MPBA SERS nanoprobe,we analyzed SA expression levels on in vitro cell lines including human cervical cancer HeLa cells（HeLa）,human liver hepatocellular carcinoma（HepG2）and mouse embryonic liver cells（BNL.CL2）and further expand to investigate the clinical breast tissue sections.Firstly,SERS detection was performed for human breast cancer and normal tissue sections and the characteristic peaks located at 1074 and 1572 cm^-1 were used for analyzing the SA levels.Then,to assess the accuracy,receiver operating characteristic（ROC）curves and its area under curve（AUC）values were analyzed.The results showed that the AUC values for peaks at 1074 and 1572 cm^-1 are 0.9419 and 0.9012,confirming the method possesses high accuracy.Besides,SERS mapping is used for further characterize the expression levels and distributions on human breast cancer and normal tissues.Drug-resistance bacteria has been become a global problem that pose great risk to human health since the abuse of the antibiotics cause the emerging of multi-drug resistance strains.The mechanisms of drug-resistance at cellular levels include intrinsically resistance and acquired resistance by increasing efflux,changing the antibiotics targets by mutation and modifying the antibiotics.Besides,once persister and biofilm formed,the resistant ability of bacteria will be enhanced more than hundreds and even thousands times.To overcome resistance,the new antibacterial agents are developing but it takes a lot of time and money.The new resistance strains will emerge after a shorter time when the new drugs were introduced to clinical application.Namely,the discovery of the new antibiotics is not keeping the pace with the emergence of the drug-resistance bacterial strains,which signifies sensitizing drug-resistance bacteria to conventional antibiotics in clinic used and developing the new strategies that can easily translated into clinic application are much meaningful.For this part,we developed an approach that sensitize MRSA to membrane-targeting antibiotics with the assistance of 460 nm light which can specifically bleach the pigment of staphyloxanthin on MRSA cell membrane.The main contents and achievement are outlines as follows:1)We proposed a strategy that 460 nm light can affect the permeability and fluidity and further enhance the antibacterial effect of membrane-targeting antibiotics to MRSA by photobleaching of staphyloxanthin the specific pigment on cell membrane.The results showed that 460 nm light can bleach Staphyloxanthin,the specific pigment on MRSA cell membrane,which can enhance the permeability and fluidity.As the classic membrane-targeting antibiotics,460 nm light can make more Daptomycin molecules insert cell membrane.By analysis,the effect between 460 nm light and Daptomycin is synergistic,and the index is below than 0.416.Furthermore,460 nm light can enhance the antibacterial effect of other kinds of membrane-active antibiotics and this method is durable for other drug-resistance S.aureus strains.2)By the method that the antibacterial effect of membrane-targeting antibiotics can be enhanced with 460 nm light,we further studied the persister and biofilm models that formed by MRSA.The results showed that this method can effectively enhance membrane-targeting antibiotics against MRSA formed biofilm and persister.Besides,we evaluated the toxicity of this method shown to in vitro human cell line and the antibacterial effect for combating the MRSA in human blood infection,which confirmed that our method showed no obvi ous toxicity to normal cells and can be effectively used for eliminating MRSA in human blood.Furthermore,we further built the MRSA-infected wound infection and explored the therapy effect of our developed method.The results indicated that our method can achieve high antibacterial effect for the wound infection in vivo.