The Application Of Alkaline Phosphatase-Induced Peptide Self-Assembly In Tumor Imaging Analyses And Cytotoxicity Studies

Self-assembly plays an important role in nature and it provides guidance for the design and construction of functional materials.Among the initiators of self-assembly,enzyme-instructed self-assembly has been applied to various life processes due to its high controllability and biocompatibility.Taking alkaline phosphatase-instructed peptide self-assembly as an example,it usually consists of two steps.Firstly substrate molecules are dephosphorylated under the catalysis of alkaline phosphatase,and secondly the dephosphorylation products self-assemble through intermolecular interactions for biomaterial preparation,biological imaging,cancer therapy,drug delivery,etc.In this dissertation,we rationally designed several new phosphate precursors based on alkaline phosphatase instructed peptide self-assembly for biological applications,such as tumor imaging analyses and cell toxicity studies.It is expected to achieve high sensitivity for early diagnosis of cancer or provide new prospects for the development of nanomedicine to treat cancers.In chapter 2 of this dissertation,we reported a NIR probe IR775-Phe-Phe-Tyr(H2PO3)-OH(1P)for PA imaging ALP activity in vitro and in tumor employing enzyme-instructed self-assembly strategy.Under the catalysis of ALP,1P was efficiently converted to IR775-Phe-Phe-Tyr-OH(1),which self-assembled into the nanoparticles 1-NPs.The formation of 1-NPs induced a 6.4-fold enhancement of the 795 nm PA signal of 1P.Using this property,we developed an assay for quantitate ALP activity in vitro.In vivo tumor PA imaging results indicated that,compared to that in the ALP inhibitor-treated control group,PA contrast in the experimental group enhanced 2.3 folds at 4 h after 1P injection.We envision that our strategy could be applied for highly sensitive diagnosis of superficial cancers over-expressing ALP in the near future.What’s more,by replacing the Phe-Phe-Tyr(H2PO3)-OH motif in 1P with other enzyme-cleavable ones,we hope that more PA probes could be developed for the precise diagnoses of their corresponding cancers in the near future.In chapter 3 of this dissertation,we rationally designed three hydrogelators and their corresponding phosphate precursors,and systematically studied how fluorination affected their self-assembling ability and cytotoxicity.The results indicated that fluorine substitution,but not trifluoromethyl substitution with more fluorine atoms,to the phenylalanine motif enhanced the self-assembling ability and cytotoxicity of the hydrogelators(or precursors).Gel 2 and Gel 2P had the highest mechanical strength among the physical and enzymatic hydrogels,respectively.Our cytotoxicity study indicated that 2,the fluorinated hydrogelator which had the best self-assembling ability,exhibited the highest cytotoxicity among the six compounds.We envision that our preliminary study of hydrogelator fluorination would provide a strategy for the development of supramolecular hydrogels with biological application values.In chapter 4 of this dissertation,we rationally designed a pyrene-peptide conjugate Py-Phe-Phe-Glu-Tyr(H2PO3)-Gly-lyso(Py-Yp-Lyso)and demonstrated its lysosome-targeting cytotoxicity on cancer cells,together with its pyrene(Py)excimer fluorescence turning "on" at 480 nm.In vitro results showed that,Py-Yp-Lyso was efficiently dephosphorylated by ALP to yield Py-Phe-Phe-Glu-Tyr-Gly-lyso(Py-Y-Lyso)which self-assembled into nanofibers.Cell experiments verified that,after taken up by HeLa cells,the excimer fluorescence of Py-Yp-Lyso assemblies was turned "on" and the assemblies specifically targeted the lysosomes,inducing LMP and ultimate cancer cell death.In vivo experiments indicated that Py-Yp-Lyso had the highest inhibition effect towards HeLa tumors among the four compounds studied.We anticipate to apply Py-Yp-Lyso to treat cancers in clinic in the future.This dissertation aims at the application of alkaline phosphatase-instructed peptide self-assembly in tumor imaging analyses and cytotoxicity studies.We hope that this strategy could provide guidance for the highly sensitive diagnosis of early cancer and the development of new anticancer nanodrugs.