Application Of Alkaline Phosphatase-Regulated Peptide Self-Assembled Hydrogel In Imaging

Molecular self-assembly refers to the spontaneous arrangement of molecules in a certain order,the phenomenon that is ubiquitous in nature.Self-assembly is a key concept in supramolecular chemistry,based primarily on non-covalent interactions(such as van der Waals forces,hydrogen bonds,electrostatic interactions,and hydrophobic interactions between molecules).Among these non-covalent interactions,the hydrophobic interaction is a result of long-term interaction of non-polar groups in water,and has a unique and important effect on the self-assembly of hydrogels(or small molecules)in water.Self-assembled hydrogel nanofibers act as a network or matrix of supramolecular hydrogels with many of the characteristics of certain biological processes in nature(such as the kinetic processes of cytoskeleton such as actin filaments and microtubules),that is a basic molecular process,usually non-covalent,three-dimensional,dynamic,adaptive,responsive,and organized.This molecular process can easily interact with,interfere with and even imitate the intracellular events of a variety of biological systems,thus promising new applications in the field of biology and biomedicine.The pathogenesis,development and sensitivity to treatment of disease are usually accompanied by abnormal activities of various biomolecules in and around the corresponding cells before obvious anatomical and physiological changes.Molecular imaging technology can accurately locate the location and expression level of these biomolecules,so as to achieve early diagnosis of related diseases,design appropriate treatment strategies,and accurately evaluate the treatment effect.In the first part of this paper,we developed a fluorescent probe P1 for quantitative detection of alkaline phosphatase(ALP)active living cells in vitro and in vivo.Under the catalytic hydrolysis of ALP,compound P1 was transformed into gel factor 1 and self-assembled into nanofibers to form hydrogels.Along with this sol-gel transition,the fluorescent emission of P1 is "turned off".Rheological tests were used to study the viscoelastic properties of the gel.Transmission electron microscopy(TEM)image indicates that the gel consists of nanofibers with an average diameter of approximately 10 nm.Quantitative analysis in vitro showed that P1 could be used to detect ALP.The linear range of ALP concentration was 0-2.8 U/mL,and the detection limit was 0.06 U/mL.Cell imaging studies showed that the fluorescence emission intensity of LoVo cells pretreated with ALP inhibitor was 1.7 times that of LoVo cells directly incubated with P1,indicating that P1 can be used to detect ALP activity in living cells.We combined "sol-gel" transformation with the fluorescence signal "off" method to provide a new strategy for real-time detection of ALP activity in vitro and in living cells.In the second part of this paper,We designed the precursor 1P containing Gd chelate,self-assembled under the regulation of alkaline phosphatase and caused the enhancement of T2-weighted magnetic resonance imaging(MRI)signal,and achieved the activity of detecting ALP in vitro and in vivo respectively.Under the catalytic hydrolysis of ALP,compound 1P was dephosphorylated to produce compound 1,which was self-assembled into nanofibers to further form hydrogels.The test results of rheological mechanics show that the gel has good mechanical properties.Cryo-TEM showed that the resulting hydrogels were networks of nanofibers about 6.5 nm in diameter.T2 images showed that the r2 value of the gel was 33.9%higher than that of 1P,which was one order of magnitude higher than that of Gd-DTPA.In vivo T2-weighted magnetic resonance imaging showed that at 9.4T,Probe 1P was applied to mice by tail vein injection,and T2-weighted magnetic resonance imaging was obtained by subcutaneous transplantation of phosphatase overexpressing HeLa tumor sites.T2 enhanced images were obtained compared with the control group.We expect that enzyme-induced Gd-peptide self-assembly may be an important complementary strategy for the development of "smart" T2 MRI probes for enzymatic activity in vivo.The work of these two parts shows that the supramolecular hydrogel assembly strategy is an effective method for constructing fluorescence or MRI analytical image probes,and it is expected to be further applied to the construction of other intelligent molecular imaging probes for biological function recognition.