Synthesis And Application Of Azoreductase-Based Near-Infrared Fluorescent Polymer Probe

Stimuli-responsive materials can undergo changes in response to slight physical or chemical stimuli in the external environment,such as light,temperature.pH and biological changes.Stimuli-responsive materials have been widely used in the fields of drug release,biological imaging and bioactive molecule carrier.Enzymes play an important role among the many stimulus,because of their high efficiency,selectivity and particularity in human body.Targeted delivery and controlled drug release can be achieved according to the specific response of enzymes.In addition to its unique optical response,azobenzene is sensitive to azoreductase produced by the microbial flflora present in the colon.Therefore,azobenzene can be applied in biotechnology as an active site of azoreductase in colon.Fluorescent probes have emerged as a kind of highly efficient and sensitive biodetector,and will have an important application in bioimaging and biosensing.As we know,the absorption,scattering and spontaneous fluorescence of visible light by organisms reduce the sensitivity of the fluorescent probe in the visible light region.The maximum absorption and emission wavelength of near-infrared fluorescent probes are between 600?950 nm,which can effectively avoid the background interference of tissues.Besides,it can infiltrate into the organism to increase its sensitivity,playing an important role in bioimaging.Near-infrared fluorescent probe can realize the recognition of enzymes,drug release monitoring and bioimaging in vivo.Compared with small molecular compounds,polymers have lower toxicity,more structural diversity,better solubility and stability.Besides,they can be assembled to prepare nanomaterials.Therefore,fluorescent polymer probes have been a research hotspot because of their unique advantages.Through the literature investigation,we found that there are few reports on the near-infrared fluorescent polymer probes based on the response of azoreductase.The current researches are not deep enough in polymers based on azoreductase.In fact,most of them focus on reduction-responsive dissociation of polymer assemblies,and there are few reports on their applications,while reports on dual-function polymers based on drug release and fluorescence detection are more rarer.Recently,our group reported an azoreductase responsive fluorescent polymer probe based on tetraphenyl ethylene,whose fluorescent emission wavelength is within the visible region.As mentioned above,the fluorescent probe in the visible region has certain defects in the application in vivo.Therefore,we focus on the near-infrared fluorescent polymer probe based on azoreductase,aiming to expand the application of azobenzene polymers in biosensing,drug carrier and other fields,broadening the variety of macromolecular fluorescent probes based on azoreductase,so as to provide necessary theoretical basis for colon-targeted drug delivery carrier and fluorescence monitoring system in vivo and fill in the blank of this research field.In this paper,we designed and synthesised a kind of near-infrared fluorescent polymer probes based on the response of azoreductase.Azobenzene and near-infrared fluorescence group(BODIPY)were insetted into the biocompatible amphiphilic block copolymer,and azobenzene endows the polymer with reduction responsiveness.Subsequently,the polymer and drug were mixed and assembled in solution to obtain the drug-loaded micelle.Since the fluorescence group is wrapped in the core of the micelle,the aggregation induced quenching(ACQ)effect leads to no fluorescence emission,that is,the fluorescence "off".However,under the reduction of azoreductase,the fracture of azo bond in the micelle leds to the decomposition of polymer into hydrophilic and hydrophobic chains,resulting in the dissociation of micelles and release of drugs.With disconnection of the polymer,the near-infrared fluorescence group was dissolved in PBS solution due to the connection to the hydrophilic chain.The fluorescence was activated and greatly enhanced,with the elimination of ACQ effect,that is,the fluorescence "on".The details are as follows:1.A small molecule fluorescent probe containing azobenzene and near-infrared fluorescent group(BODIPY)was designed and synthesized firstly.After that,RAFT polymerization was conducted to obtain a well-structured amphiphilic block copolymer(PBAPMA-b-POEGMA).The micromolecule fluorescent probe was then attached to the side chain of the polymer by Michael addition to obtain the amphiphilic near-infrared fluorescent polymer probe(P(BAPMA-co-APMA-co-RAPMA)-b-POEGMA).The structure of the polymer was characterized by 1H NMR,GPC and UV-Vis.The micelles were obtained through the subsequently self-assembled of polymers in PBS solution,and the morphology of micelles was characterized by transmission electron microscopy(TEM),indicating that a stable spherical micelle with a diameter of about 30 nm could be formed in PBS buffer solution.Then the residual amino groups at the interface between the shell and the core were cross-linked by cross-linking agent with double bond,which made the micelles more stable.In order to simulate the environment in colon,the sodium disulfite(Na2S2O4)was added at 37? under anaerobic conditions to trigger the reduction of the micelle.TEM,fluorescence spectrophotometer and UV/Vis spectrophotometer were used to characterize the morphology of micelles,the fluorescence and UV-Vis absorption changes of micelle solutions before and after reduction.The results show that the polymer has redox triggered fluorescence.2.A small molecule fluorescent probe based on azobenzene and near-infrared fluorescence group(BODIPY)was first designed and synthesized,then insetted into the main chain of the amphiphilic block copolymer PEG398-alAB6Azo-PLA144.connecting the hydrophilic and hydrophobic segments by ROP and CuAAC(Cu-catalyzed azide/alkyne cycloaddition)"Click" reaction.The structure of the polymer was characterized by 1H NMR,GPC and FT-IR.The polymer PEG398-alAB6Azo-PLA144 was subsequently self-assembled in DMSO mixed with PBS(pH=7.4)to obtain the polymer assembly with fluorescent "off".The morphology,size and particle size distribution of the assembly were characterized by TEM and dynamic light scattering(DLS).The results showed that a stable spherical micelle with a diameter of about 35 nm could be formed in PBS solution(pH 7.4).The colonic environment was further simulated,and azoreductase along with NADPH were used to enzymatic hydrolyze the micelles in PBS solution at 37 C.The fluorescence "on/off" behavior was studied by fluorescence spectrophotometer,the fracture of azobenzene was observed by UV/Vis spectrophotometer,and the reduced micelle morphology,size and particle size distribution were fully characterized by TEM and DLS.The results showed that azobenzene could be completely broken in the presence of azoreductase,the assembly was slowly dissociated,and the fluorescence was gradually activated near 710 nm.MTT cytotoxicity tests were conducted on the empty micelle before and after enzymatic hydrolysis using normal cell L929 and cancer cell Hela.The results showed that the PEG-alAB6Azo-PLA empty micelle and the polymer fragments with exposed aniline after enzymatic hydrolysis were non-toxic and had good biocompatibility.The drug loaded micelles with fluorescence "off" were subsequently assembled by PEG398-alAB6Azo-PLA144 and model drug DOX in PBS solution,then the azoreductase responsiveness of drug loaded micelle was investigated in a simulated environment of colon at 37?.The fluorescence intensity and drug release was monitored by the fluorescence spectrophotometer,the fracture progress of azobenzene were characterized by UV-Vis,the micelle morphology was observed during the reduction by TEM,and particle size and distribution were characterized by DLS before and after the reduction.The results showed that the drug-loaded micelles were dissociated gradually in the presence of azoreductase,which could realize sustained release of drugs and synchronous enhancement of near-infrared fluorescence.In a certain degree,the drug release could be tracked by monitoring the change of fluorescence intensity.